Patent Publication Number: US-2018031562-A1

Title: Cancer biomarkers

Description:
The present invention relates generally to prostate cancer biomarkers and to methods of screening for prostate cancer. Such methods involve determining the level of certain biomarkers which are indicative of prostate cancer in a subject. 
     Prostate cancer is a global health problem. It represents 12% of all cancer cases worldwide, and it is the second most commonly diagnosed cancer in men (Baade P D, Youlden D R, &amp; Krnjacki L J (2009) International epidemiology of prostate cancer: geographical distribution and secular trends.  Mol Nutr. Food Res.,  53, 171-184). Prostate specific antigen (PSA) has been used for nearly three decades as a biomarker for prostate cancer and is still a useful marker for prostate cancer after diagnosis. However, the serum PSA test lacks sensitivity and specificity, and this has resulted in prostate cancer overdiagnosis and overtreatment (Welch H G &amp; Albertsen P C (2009) Prostate cancer diagnosis and treatment after the introduction of prostate-specific antigen screening: 1986-2005.  J. Natl. Cancer Inst.,  101, 1325-1329). Recently, the U.S. Preventive Services Task force decided to recommend against the use of this biomarker for prostate cancer screening (Moyer V A (2012) Screening for prostate cancer: U.S. Preventive Services Task Force recommendation statement.  Ann. Intern. Med.,  157, 120-134). 
     PSA also suffers from a high rate of false negatives, as it has been reported that as many as 15% of patients with clinically significant prostate cancer (Thompson et al., 2003,  New England Journal of Medicine;  349(3): 215-224) had normal PSA levels. Thus, the PSA test is inadequate with respect to both sensitivity and specificity. This illustrates the need for a diagnostic test that would reduce the number of both false positives and false negatives and improve early diagnosis. 
     A rise in PSA levels combined with a positive digital rectal exam (DRE) typically leads to referral of the patient to a urologist for a biopsy to confirm diagnosis of prostate cancer, as well as determine its grade. Since it is easy to miss a small cancer tissue within the prostate consisting of otherwise healthy tissue, many samples from different regions of the prostate are typically collected at each biopsy procedure. Nevertheless, sampling errors can still result in cancer being missed in up to 25% of cases, necessitating repeated biopsy procedures in case of negative results, with the associated discomforts and risks. There is thus also a need for supplementary non-invasive tests that may be administered after a negative biopsy to determine the need for repeat biopsies. 
     Examination of biopsies by a pathologist is used to determine the grade (Gleason score) of the cancer. The Gleason score is used in combination with information regarding the localization of the tumor within and around the prostate to determine the Stage I-IV, where IV is the most aggressive. Following diagnosis of prostate cancer, management decisions are currently based on numerous risk stratification systems that are generally based on different threshold and weighting of three key parameters to indicate high-risk disease: PSA levels, Gleason score, and clinical stage of the disease. Various classification guidelines based on these parameters are in existence, and may give drastically different results, leading to possible over- or under-treatment (Buck &amp; Chughtai, 2014,  B. Nat. Rev. Urol.  11:256-257). There is thus also a clear unmet need for biomarkers that may improve risk stratification. 
     Since prostate cancer is in many cases a slowly progressing disease, it is increasingly recommended that very low-risk patients do not immediately seek treatment (National Comprehensive Cancer Network guidelines, 2012), but are subject to watchful waiting or active surveillance. Active surveillance requires frequent testing involving biopsies, which are invasive, and PSA screening, which is often utilized, although its benefits and accuracy are both hotly disputed. With better monitoring tools, patients and doctors will be more comfortable choosing conservative management and postponing treatment. 
     What is needed in the art are new methods of screening for prostate cancer. Such methods may be useful for assessing whether a subject qualifies for a first biopsy, reducing false negative biopsies (decision on whether to perform additional biopsies), distinguishing between indolent and aggressive cancer (decision between active surveillance and treatment), and monitoring of patients under active surveillance. Preferably such methods would be non-invasive and performed on readily obtainable samples. The identification of novel biomarkers for prostate cancer may potentially have clinical implications for a large number of patients. 
     The present inventors have identified certain polypeptides (proteins) that are differentially expressed in urinary exosomes from prostate cancer patients in comparison to control subjects. These differentially expressed polypeptides act as biomarkers for prostate cancer and thus are useful in screening for prostate cancer in subjects. Such biomarkers may also be used in methods of assessing whether or not a subject qualifies for first biopsy, reducing false negative biopsies (decision on whether to perform additional biopsies), distinguishing between indolent and aggressive cancer (decision between active surveillance and treatment), and monitoring of patients under active surveillance. 
     Thus, in one aspect the present invention provides a method of screening for prostate cancer in a subject, said method comprising 
     determining the level in a sample of one or more polypeptides selected from the group consisting of:
         Transmembrane protein 256, Adipogenesis regulatory factor, Ragulator complex protein LAMTOR1, Plastin-2, Ras-related protein Rab-2A, Ras-related protein Rab-3B, Ras-related protein Rab-3D, Ras-related protein Rab-7a, V-type proton ATPase 16 kDa proteolipid subunit, Metalloreductase STEAP4, Protein DJ-1, Protein S100-P, Synaptotagmin-like protein 4, ADP-ribosylation factor-like protein 8B, Proton myo-inositol cotransporter, Ras-related protein Rab-6A, Tetraspanin-6, Claudin-10, Claudin-2, Claudin-3, GDP-mannose 4.6 dehydratase, Glucosamine-6-phosphate isomerase 1, Lysosome membrane protein 2, Major facilitator superfamily domain-containing protein 12, Melanophilin, Sepiapterin reductase, Thioredoxin domain-containing protein 17, 3-hydroxybutyrate dehydrogenase type 2, Calmodulin, Carboxypeptidase Q, Flotillin-2, Galectin-3-binding protein, P2X purinoceptor 4, Protein dopey-2, Protein S100-A6, Ras-related protein Rab-35, Serine/threonine-protein phosphatase 2A catalytic subunit alpha iso form, 2′-deoxynucleoside 5′-phosphate N-hydrolase 1, Acid ceramidase, Calbindin, CD59 glycoprotein, CD81 antigen, Cytochrome b561, Enolase-phosphatase E1, Golgi phosphoprotein 3, Nicastrin, Probable serine carboxypeptidase CPVL, Ragulator complex protein LAMTORS, Ras-related protein Rab-27B, Secretory carrier-associated membrane protein 2, Spermine synthase, S-phase kinase-associated protein 1, Transmembrane 7 superfamily member 3, Tumor protein D52, Ubiquitin-conjugating enzyme E2 variant 2, UDP-glucose 6-dehydrogenase, Zinc-alpha-2-glycoprotein, Glycerophosphodiester phosphodiesterase domain-containing protein 3, EF-hand domain-containing protein D2, Ras-related protein Rab-14, Omega-amidase NIT2, Alpha-actinin-1, Monocarboxylate transporter 5, Ras-related protein Rab-12, Ras-related protein Rab-8A, Transmembrane protein 63A, Beta-2-microglobulin, V-type proton ATPase subunit d 1, Lipid phosphate phosphohydrolase 1, Integral membrane protein GPR155, 14-3-3 protein sigma, E3 ubiquitin-protein ligase LRSAM1, HLA class II histocompatibility antigen. DM alpha chain, Ras-related protein Rab-9A, Src substrate cortactin, Aquaporin-7, Gamma-synuclein, 14-3-3 protein theta, Aspartate aminotransferase. cytoplasmic, Chloride intracellular channel protein 3, Destrin, GTPase HRas, Prostaglandin reductase 2, T-complex protein 1 subunit epsilon, Inter-alpha-trypsin inhibitor heavy chain H4, Aldehyde dehydrogenase family 1 member A3, Annexin A3, Battenin, Cathepsin D, N(G).N(G)-dimethylarginine dimethylaminohydrolase 1, Neural proliferation differentiation and control protein 1, Proactivator polypeptide, Prostate-specific antigen, Protein lifeguard 3, Protein Niban, Protein spinster homolog 1, Ragulator complex protein LAMTOR2, Ragulator complex protein LAMTOR3, Synaptotagmin-7, Transmembrane protein 106B, Unconventional myosin-Vc, Vesicle-associated membrane protein 2, V-type proton ATPase subunit F, A-kinase anchor protein 7 isoforms alpha and beta, Arylsulfatase F, C-Jun-amino-terminal kinase-interacting protein 4, Deleted in malignant brain tumors 1 protein, Glia maturation factor beta, Glutamate carboxypeptidase 2, Glutathione synthetase, Hippocalcin-like protein 1, Myristoylated alanine-rich C-kinase substrate, NAD(P)H-hydrate epimerase, Napsin-A, Phosphoacetylglucosamine mutase, Probable phospholipid-transporting ATPase IA, Quinone oxidoreductase, Retinol-binding protein 5, Serine incorporator 2, Solute carrier family 15 member 1, Sorcin, Sucrase-isomaltase. Intestinal, Actin-related protein ⅔ complex subunit 4, Actin-related protein ⅔ complex subunit 5, Adenine phosphoribosyltransferase, Costars family protein ABRACL, Ig alpha-1 chain C region, Interferon-induced transmembrane protein 3, Lactotransferrin, Purine nucleoside phosphorylase, Syntaxin-binding protein 4, Tetraspanin-8, Vacuolar protein-sorting-associated protein 25, Voltage-dependent anion-selective channel protein 1, Collagen alpha-1(VI) chain, Signal transducing adapter molecule 1, CD9 antigen, Flotillin-1, Grancalcin, Mannose-1-phosphate guanyltransferase beta, Proteasome subunit alpha type-7, Ras-related protein Rab-18, Vacuolar protein sorting-associated protein 37C, Signal transducing adapter molecule 2, Pancreatic secretory granule membrane major glycoprotein GP2, 1-phosphatidylinosito14.5-bisphosphate phosphodiesterase gamma-2, Lysosome-associated membrane glycoprotein 2, Cysteine-rich C-terminal protein 1, Plasma serine protease inhibitor, Tyrosine-protein kinase Lck, Ragulator complex protein LAMTOR4, Secretory carrier-associated membrane protein 1, Peptidyl-prolyl cis-trans isomerase FKBP1A, Transmembrane protein 176A, Thymosin beta-4, Haloacid dehalogenase-like hydrolase domain-containing protein 2, Cell division control protein 42 homolog, Ras-related protein Rab-17, Chloride intracellular channel protein 6, Choline transporter-like protein 4, Flavin reductase (NADPH), Ras-related protein Rab-10, Heme-binding protein 2, Fatty acid-binding protein. epidermal, Small integral membrane protein 5, Lysosomal-associated transmembrane protein 4A, Phytanoyl-CoA dioxygenase domain-containing protein 1, Proteasome subunit alpha type-5, Calmodulin-like protein 3, Presenilin-1, Ribosyldihydronicotinamide dehydrogenase [quinone], Translationally-controlled tumor protein, Lysosome-associated membrane glycoprotein 1, ADP-ribosyl cyclase 1, Myotrophin, Dynein light chain 2. cytoplasmic, Dihydropteridine reductase, Nicotinate-nucleotide pyrophosphorylase [carboxylating], Cell cycle control protein 50A, Phosphomevalonate kinase, Eukaryotic translation initiation factor 4H, Protein tweety homo log 3, Sodium-dependent phosphate transport protein 2B, Ig lambda-2 chain C regions, Cellular retinoic acid-binding protein 2, Protein CutA, Proteasome subunit alpha type-4, Solute carrier family 35 member F6, Delta-aminolevulinic acid dehydratase, L-xylulose reductase, Uroplakin-1a, Cornifin-A, Zinc finger protein 185, Transmembrane protein 8A, Prenylcysteine oxidase 1, Lysozyme C, Paralemmin-1, Carcinoembryonic antigen-related cell adhesion molecule 6, Sodium/glucose cotransporter 1, Prostaglandin reductase 1, Protein S100-A9, MICAL-like protein 1, Alpha/beta hydrolase domain-containing protein 14B, Aquaporin-2, Glutathione S-transferase P, Probable almitoyltransferase ZDHHC1, Ras-related protein Rab-8B, Transmembrane protease serine 2, Ras-related protein Rab-1B, Ras-related protein Rab-1A, Ras-related protein Rab-43, Synaptogyrin-2, HLA class II histocompatibility antigen. DRB1-15 beta chain, Lysosomal protein NCU-G1, Calcium-binding protein 39, Dynamin-2, CDC42 small effector protein 2, Ferritin heavy chain, Solute carrier family 35 member F2, Probable hydrolase PNKD, Cathepsin Z, Tubulin beta-2B chain, Thiosulfate sulfurtransferase/rhodanese-like domain-containing protein 1, Mitogen-activated protein kinase 1, Alcohol dehydrogenase class-3, Low molecular weight phosphotyrosine protein phosphatase, Annexin A4, Septin-2, Glutathione S-transferase Mu 3, Protein BRICK1, Proteasome subunit beta type-2, Ubiquitin- conjugating enzyme E2 K, Protein S100-A1, Microtubule-associated protein 1A, Glutathione S-transferase Mu 1, Matrix metalloproteinase-24, Small integral membrane protein 22, Heparan-alpha-glucosaminide N-acetyltransferase, Specifically androgen-regulated gene protein, Abl interactor 1, Uncharacterized protein C6orf132, ADP-ribosylation factor 5, Isocitrate dehydrogenase [NADP] cytoplasmic, Glycolipid transfer protein, Tropomyosin alpha-4 chain and Natural resistance-associated macrophage protein 2;   wherein said sample comprises urinary exosomes and wherein said sample has been obtained from said subject;   wherein an increased level in said sample of one or more of said polypeptides selected from the group consisting of Transmembrane protein 256, Adipogenesis regulatory factor, Ragulator complex protein LAMTOR1, Plastin-2, Ras-related protein Rab-2A, Ras-related protein Rab-3B, Ras-related protein Rab-3D, Ras-related protein Rab-7a, V-type proton ATPase 16 kDa proteolipid subunit, Metalloreductase STEAP4, Protein DJ-1, Protein S100-P, Synaptotagmin-like protein 4, ADP-ribosylation factor-like protein 8B, Proton myo-inositol cotransporter, Ras-related protein Rab-6A, Tetraspanin-6, Claudin-10, Claudin-2, Claudin-3, GDP-mannose 4.6 dehydratase, Glucosamine-6-phosphate isomerase 1, Lysosome membrane protein 2, Major facilitator superfamily domain-containing protein 12, Melanophilin, Sepiapterin reductase, Thioredoxin domain-containing protein 17, 3-hydroxybutyrate dehydrogenase type 2, Calmodulin, Carboxypeptidase Q, Flotillin-2, Galectin-3-binding protein, P2X purinoceptor 4, Protein dopey-2, Serine/threonine-protein phosphatase 2A catalytic subunit alpha iso form, 2′-deoxynucleoside 5′-phosphate N-hydrolase 1, Acid ceramidase, Calbindin, CD81 antigen, Cytochrome b561, Enolase-phosphatase E1, Golgi phosphoprotein 3, Nicastrin, Probable serine carboxypeptidase CPVL, Ragulator complex protein LAMTORS, Ras-related protein Rab-27B, Secretory carrier-associated membrane protein 2, Spermine synthase, S-phase kinase-associated protein 1, Transmembrane 7 superfamily member 3, Tumor protein D52, Ubiquitin-conjugating enzyme E2 variant 2, UDP-glucose 6-dehydrogenase, Zinc-alpha-2-glycoprotein, Glycerophosphodiester phosphodiesterase domain-containing protein 3, EF-hand domain-containing protein D2, Ras-related protein Rab-14, Omega-amidase NIT2, Alpha-actinin-1, Monocarboxylate transporter 5, Ras-related protein Rab-12, Ras-related protein Rab-8A, Transmembrane protein 63A, V-type proton ATPase subunit d 1, Lipid phosphate phosphohydrolase 1, Integral membrane protein GPR155, E3 ubiquitin-protein ligase LRSAM1, HLA class II histocompatibility antigen. DM alpha chain, Ras-related protein Rab-9A, Aquaporin-7, Gamma-synuclein, Aspartate aminotransferase. cytoplasmic, Chloride intracellular channel protein 3, Destrin, GTPase HRas, Prostaglandin reductase 2, T-complex protein 1 subunit epsilon, Aldehyde dehydrogenase family 1 member A3, Annexin A3, Battenin, Cathepsin D, N(G).N(G)-dimethylarginine dimethylaminohydrolase 1, Neural proliferation differentiation and control protein 1, Proactivator polypeptide, Prostate-specific antigen, Protein lifeguard 3, Protein Niban, Protein spinster homolog 1, Ragulator complex protein LAMTOR2, Ragulator complex protein LAMTOR3, Synaptotagmin-7, Transmembrane protein 106B, Unconventional myosin-Vc, Vesicle-associated membrane protein 2, V-type proton ATPase subunit F, A-kinase anchor protein 7 isoforms alpha and beta, Arylsulfatase F, C-Jun-amino-terminal kinase-interacting protein 4, Deleted in malignant brain tumors 1 protein, Glia maturation factor beta, Glutamate carboxypeptidase 2, Glutathione synthetase, Hippocalcin-like protein 1, NAD(P)H-hydrate epimerase, Napsin-A, Phosphoacetylglucosamine mutase, Probable phospholipid-transporting ATPase IA, Quinone oxidoreductase, Retinol-binding protein 5, Serine incorporator 2, Solute carrier family 15 member 1, Sorcin, Sucrase-isomaltase. Intestinal, Actin-related protein ⅔ complex subunit 4, Actin-related protein ⅔ complex subunit 5, Adenine phosphoribosyltransferase, Costars family protein ABRACL, Ig alpha-1 chain C region, Interferon-induced transmembrane protein 3, Lactotransferrin, Purine nucleoside phosphorylase, Syntaxin-binding protein 4, Vacuolar protein-sorting-associated protein 25, Voltage-dependent anion-selective channel protein 1, CD9 antigen, Flotillin-1, Grancalcin, Mannose-1-phosphate guanyltransferase beta, Proteasome subunit alpha type-7, Ras-related protein Rab-18, Vacuolar protein sorting-associated protein 37C, Pancreatic secretory granule membrane major glycoprotein GP2, Lysosome-associated membrane glycoprotein 2, Ragulator complex protein LAMTOR4, Secretory carrier-associated membrane protein 1, Peptidyl-prolyl cis-trans isomerase FKBP1A, Transmembrane protein 176A, Thymosin beta-4, Haloacid dehalogenase-like hydrolase domain-containing protein 2, Cell division control protein 42 homolog, Ras-related protein Rab-17, Chloride intracellular channel protein 6, Choline transporter-like protein 4, Flavin reductase (NADPH), Ras-related protein Rab-10, Heme-binding protein 2, Fatty acid-binding protein. epidermal, Small integral membrane protein 5, Lysosomal-associated transmembrane protein 4A, Phytanoyl-CoA dioxygenase domain-containing protein 1, Proteasome subunit alpha type-5, Calmodulin-like protein 3, Presenilin-1, Ribosyldihydronicotinamide dehydrogenase [quinone], Translationally-controlled tumor protein, Lysosome-associated membrane glycoprotein 1, ADP-ribosyl cyclase 1, Myotrophin, Dynein light chain 2. cytoplasmic, Dihydropteridine reductase, Nicotinate-nucleotide pyrophosphorylase [carboxylating], Cell cycle control protein 50A, Eukaryotic translation initiation factor 4H, Protein tweety homolog 3, Sodium-dependent phosphate transport protein 2B, Ig lambda-2 chain C regions, Cellular retinoic acid-binding protein 2, Protein CutA, Proteasome subunit alpha type-4, Solute carrier family 35 member F6, Delta-aminolevulinic acid dehydratase, L-xylulose reductase, Uroplakin-1a, Comifin-A, Zinc finger protein 185, Transmembrane protein 8A, Prenylcysteine oxidase 1, Lysozyme C, Paralemmin-1, Carcinoembryonic antigen-related cell adhesion molecule 6, Sodium/glucose cotransporter 1, Prostaglandin reductase 1, Protein S100-A9, MICAL-like protein 1, Aquaporin-2, Glutathione S-transferase P, Ras-related protein Rab-8B, Transmembrane protease serine 2, Ras-related protein Rab-1B, Ras-related protein Rab-1A, Ras-related protein Rab-43, Synaptogyrin-2, HLA class II histocompatibility antigen. DRB1-15 beta chain, Lysosomal protein NCU-G1, CDC42 small effector protein 2, Ferritin heavy chain, Solute carrier family 35 member F2,Probable hydrolase PNKD, Cathepsin Z, Tubulin beta-2B chain, Mitogen-activated protein kinase 1, Alcohol dehydrogenase class-3, Low molecular weight phosphotyrosine protein phosphatase, Annexin A4, Septin-2, Glutathione S-transferase Mu 3, Protein BRICK1, Proteasome subunit beta type-2, Ubiquitin- conjugating enzyme E2 K, Protein S100-A1, Microtubule-associated protein 1A, Glutathione S-transferase Mu 1, Small integral membrane protein 22, Heparan-alpha-glucosaminide N-acetyltransferase, Specifically androgen-regulated gene protein, Abl interactor 1, Uncharacterized protein C6orf132, Isocitrate dehydrogenase [NADP] cytoplasmic, Glycolipid transfer protein, Tropomyosin alpha-4 chain and Natural resistance-associated macrophage protein 2 in comparison to a control level is indicative of prostate cancer in said subject; and/or   wherein a decreased level in said sample of one or more of said polypeptides selected from the group consisting of Protein S100-A6, Ras-related protein Rab-35, CD59 glycoprotein, Beta-2-microglobulin, 14-3-3 protein sigma, Src substrate cortactin, 14-3-3 protein theta, Inter-alpha-trypsin inhibitor heavy chain H4, Myristoylated alanine-rich C-kinase substrate, Tetraspanin-8, Collagen alpha-1(VI) chain, Signal transducing adapter molecule 1, Signal transducing adapter molecule 2, 1-phosphatidylinosito14.5-bisphosphate phosphodiesterase gamma-2, Cysteine-rich C-terminal protein 1, Plasma serine protease inhibitor, Tyrosine-protein kinase Lck, Phosphomevalonate kinase, Alpha/beta hydrolase domain-containing protein 14B, Probable almitoyltransferase ZDHHC1, Calcium-binding protein 39, Dynamin-2, Thiosulfate sulfurtransferase/rhodanese-like domain-containing protein 1, Matrix metalloproteinase-24 and ADP-ribosylation factor 5 in comparison to a control level is indicative of prostate cancer in said subject.       

     In one embodiment, the method comprises determining the level in a sample of one or more polypeptides selected from the group consisting of:
         Transmembrane protein 256, Adipogenesis regulatory factor, Ragulator complex protein LAMTOR1, Plastin-2, Ras-related protein Rab-2A, Ras-related protein Rab-3B, Ras-related protein Rab-3D, Ras-related protein Rab-7a, V-type proton ATPase 16 kDa proteolipid subunit, Metalloreductase STEAP4, Protein DJ-1, Protein S100-P, Synaptotagmin-like protein 4, ADP-ribosylation factor-like protein 8B, Proton myo-inositol cotransporter, Ras-related protein Rab-6A, Tetraspanin-6, Claudin-10, Claudin-2, Claudin-3, GDP-mannose 4.6 dehydratase, Glucosamine-6-phosphate isomerase 1, Lysosome membrane protein 2, Major facilitator superfamily domain-containing protein 12, Melanophilin, Sepiapterin reductase, Thioredoxin domain-containing protein 17, 3-hydroxybutyrate dehydrogenase type 2, Calmodulin, Carboxypeptidase Q, Flotillin-2, Galectin-3-binding protein, P2X purinoceptor 4, Protein dopey-2, Protein S100-A6, Ras-related protein Rab-35, Serine/threonine-protein phosphatase 2A catalytic subunit alpha iso form, 2′-deoxynucleoside 5′-phosphate N-hydrolase 1, Acid ceramidase, Calbindin, CD59 glycoprotein, CD81 antigen, Cytochrome b561, Enolase-phosphatase E1, Golgi phosphoprotein 3, Nicastrin, Probable serine carboxypeptidase CPVL, Ragulator complex protein LAMTORS, Ras-related protein Rab-27B, Secretory carrier-associated membrane protein 2, Spermine synthase, S-phase kinase-associated protein 1, Transmembrane 7 superfamily member 3, Tumor protein D52, Ubiquitin-conjugating enzyme E2 variant 2, UDP-glucose 6-dehydrogenase, Zinc-alpha-2-glycoprotein and Glycerophosphodiester phosphodiesterase domain-containing protein 3.       

     In one embodiment, the method comprises determining the level in a sample of one or more polypeptides selected from the group consisting of:
         Transmembrane protein 256, Adipogenesis regulatory factor, Ragulator complex protein LAMTOR1, Plastin-2, Ras-related protein Rab-2A, Ras-related protein Rab-3B, Ras-related protein Rab-3D, Ras-related protein Rab-7a, V-type proton ATPase 16 kDa proteolipid subunit, Metalloreductase STEAP4, Protein DJ-1, Protein S100-P, Synaptotagmin-like protein 4, ADP-ribosylation factor-like protein 8B, Proton myo-inositol cotransporter, Ras-related protein Rab-6A and Tetraspanin-6.       

     In one embodiment, the method comprises determining the level in a sample of one or more polypeptides selected from the group consisting of:
         Transmembrane protein 256, Adipogenesis regulatory factor, Ragulator complex protein LAMTOR1, Plastin-2, Ras-related protein Rab-2A, Ras-related protein Rab-3B, Ras-related protein Rab-3D, Ras-related protein Rab-7a, V-type proton ATPase 16 kDa proteolipid subunit, Metalloreductase STEAP4, Protein DJ-1, Protein S100-P, Synaptotagmin-like protein 4, ADP-ribosylation factor-like protein 8B, Proton myo-inositol cotransporter, Ras-related protein Rab-6A, Tetraspanin-6, Claudin-3, Protein S100-A6 and UDP-glucose 6-dehydrogenase.       

     In one embodiment, the levels of the polypeptides described herein are determined by mass spectrometry. 
     In one embodiment, the levels of the polypeptides described herein are determined by an immunoassay, such as, but not limited to, Western blotting and ELISA. 
     In one embodiment, the method comprises determining the level in a sample of one or more polypeptides selected from the group consisting of: Transmembrane protein 256, Ragulator complex protein LAMTOR1, Ras-related protein Rab-3B, Flotillin-1, Flotillin-2 and Protein DJ-1. In one embodiment, the method comprises determining the level in a sample of one or more polypeptides selected from the group consisting of: Transmembrane protein 256, Ragulator complex protein LAMTOR1, Ras-related protein Rab-3B, Flotillin-1 and Flotillin-2. In some such embodiments the level in a sample is determined by Western blotting or another immunoassay based method, including ELISA. 
     In one embodiment, the method comprises determining the level in a sample of one or more polypeptides selected from the group consisting of:
         Transmembrane protein 256, Adipogenesis regulatory factor, Ragulator complex protein LAMTOR1, Vesicle-associated membrane protein 2, V-type proton ATPase 16 kDa proteolipid subunit, Acid ceramidase, Prenylcysteine oxidase 1, Sorcin, Grancalcin, Ras-related protein Rab-7a, Tetraspanin-6, 3-hydroxybutyrate dehydrogenase type 2, EF-hand domain-containing protein D2, Flotillin-2, Ras-related protein Rab-3D, Adenine phosphoribosyltransferase, Calmodulin, Protein DJ-1, Retinol-binding protein 5, Ubiquitin-conjugating enzyme E2 variant 2, S-phase kinase-associated protein 1, ADP-ribosylation factor-like protein 8B, Cytochrome b561, GDP-mannose 4.6 dehydratase, Matrix metalloproteinase-24, CD59 glycoprotein, Claudin-2, Glutathione synthetase, Costars family protein ABRACL, Low molecular weight phosphotyrosine protein phosphatase, A-kinase anchor protein 7 iso forms alpha and beta, Actin-related protein ⅔ complex subunit 4, 2′-deoxynucleoside 5′-phosphate N-hydrolase 1, Dihydropteridine reductase, Ras-related protein Rab-3B, Vacuolar protein-sorting-associated protein 25, Cathepsin D, L-xylulose reductase, Mannose-1-phosphate guanyltransferase beta, Napsin-A, Purine nucleoside phosphorylase, Prostaglandin reductase 1, Ras-related protein Rab-2A, Protein S100-P, Small integral membrane protein 22, Synaptotagmin-7, Tetraspanin-8, Annexin A4, CD81 antigen, Flotillin-1, Interferon-induced transmembrane protein 3, Nicastrin, Lipid phosphate phosphohydrolase 1, Ras-related protein Rab-18, Transmembrane protein 63A, Transmembrane protease serine 2 and UDP-glucose 6-dehydrogenase.   These are polypeptides (proteins) which have a combined sensitivity and specificity of at least 160% (as set forth in Table 6 herein).       

     In one embodiment, the method comprises determining the level in a sample of one or more polypeptides selected from the group consisting of:
         Transmembrane protein 256, Adipogenesis regulatory factor, Ragulator complex protein LAMTOR1, Vesicle-associated membrane protein 2, V-type proton ATPase 16 kDa proteolipid subunit, Acid ceramidase, Prenylcysteine oxidase 1, Sorcin, Grancalcin, Ras-related protein Rab-7a and Tetraspanin-6. These are polypeptides (proteins) which have a combined sensitivity and specificity of at least 170% (as set forth in Table 6 herein).       

     In another embodiment, the method comprises determining the level in a sample of one or more polypeptides set forth in Table 6 as having a combined sensitivity and specificity of at least 175% or 180%. 
     In another embodiment, the method comprises determining the level in a sample of one or more polypeptides selected from the group consisting of: 
     Vesicle-associated membrane protein 2, Prenylcysteine oxidase 1, Sorcin and Grancalcin. 
     In another embodiment, the method comprises determining the level in a sample of one or more polypeptides selected from the group consisting of: 
     Transmembrane protein 256, Ragulator complex protein LAMTOR1, V-type proton ATPase 16 kDa proteolipid subunit, Synaptotagmin-like protein 4, Claudin-3, Protein S100-A6, UDP-glucose 6-dehydrogenase, Adipogenesis regulatory factor, Ras-related protein Rab-2A, Ras-related protein Rab-3B, Ras-related protein Rab-7a, Protein DJ-1, Tetraspanin-6, Ras-related protein Rab-3D, Protein S100-P, Proton myo-inositol cotransporter, Plastin-2, Metalloreductase STEAP4, ADP-ribosylation factor-like protein 8B, Ras-related protein Rab-6A, Vesicle-associated membrane protein 2, Prenylcysteine oxidase 1, Sorcin and Grancalcin. 
     In one embodiment, the method comprises determining the level in a sample of one or more polypeptides that are identified in Table 2 herein as having a “Validated iBAQ ratio PAT:CTR” of at least 1.75 (e.g. at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 40). 
     In one embodiment, the method comprises determining the level in a sample of one or more polypeptides that are identified in Table 2 herein as having a “Validated iBAQ ratio PAT:CTR” of at least 1 (or more than 1). In another embodiment, the method comprises determining the level in a sample of one or more polypeptides that are identified in Table 2 herein as having a “Validated iBAQ ratio PAT:CTR” of less than 1. 
     In one embodiment, the method comprises determining the level in a sample of one or more polypeptides that are referred to above as being indicative of prostate cancer when their level is increased. 
     In another embodiment, the method comprises determining the level in a sample of one or more polypeptides that are referred to above as being indicative of prostate cancer when their level is decreased. 
     In some embodiments, the method comprises determining the level in a sample of one or more polypeptides (proteins) that are identified in Table 2 or Table 3 herein as having a “sensitivity” of at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90%. In one embodiment, the level of a polypeptide (protein) identified in Table 2 herein as having a “sensitivity” of more than 40% is determined. In one embodiment, the level of a polypeptide (protein) identified in Table 2 herein as having a “sensitivity” of more than 50% is determined. In a preferred embodiment, the level of a polypeptide (protein) identified in Table 2 herein as having a “sensitivity” of more than 60% is determined. In another preferred embodiment, the level of a polypeptide (protein) identified in Table 2 herein as having a “sensitivity” of more than 70% is determined. In another preferred embodiment, the level of a polypeptide (protein) identified in Table 2 herein as having a “sensitivity” of more than 80% is determined. 
     In another embodiment, the method comprises determining the level in a sample of one or more polypeptides (proteins) that are identified in Table 2 herein as being detected in the validation study (validation analysis). 
     In another embodiment, the method comprises determining the level in a sample of one or more polypeptides (proteins) that are identified in Table 2 herein as being significantly altered in the validation study. 
     The Example herein describes certain preferred biomarkers that meet the following four criteria (see Table 2): (1) detected in validation study, (2) significantly altered in validation study, (3) sensitivity of above 40% and (4) ratio PAT versus CTR above 1.75. In relation to criteria (4), it is biomarkers whose level is increased in prostate cancer patients (samples) versus control that can have a PAT (patient) versus CTR (control) ratio of above 1.75. Analogously, for biomarkers whose level is decreased in prostate cancer patients (samples) versus control, an analogous criteria (4) may be applied, in which there is at least 1.75 times less expression of the biomarker in PAT versus CTR. In certain embodiments, the determination of the level of one or more polypeptides which meet (pass) all four of these criteria and which have a sensitivity of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% is preferred. In some embodiments , the determination of the level of one or more polypeptides which meet (pass) all four of these criteria and which have a sensitivity of at least 60% (e.g. at least 65%) is preferred. In some embodiments, the determination of the level of one or more polypeptides which meet (pass) all four of these criteria and which have a sensitivity of at least 75% is preferred. In some embodiments , the determination of the level of one or more polypeptides which meet (pass) all four of these criteria and which have a sensitivity of at least 80% is preferred. 
     In a preferred embodiment, the method comprises determining the level of Transmembrane protein 256. 
     In one embodiment, the method comprises determining the level of Ragulator complex protein LAMTOR1. 
     In one embodiment, the method comprises determining the level of V-type proton ATPase 16 kDa proteolipid subunit. 
     In one embodiment, the method comprises determining the level of Synaptotagmin-like protein 4. 
     In one embodiment, the method comprises determining the level of Claudin-3. 
     In one embodiment, the method comprises determining the level of Protein S100-A6. 
     In one embodiment, the method comprises determining the level of UDP-glucose 6-dehydrogenase. 
     In one embodiment, the method comprises determining the level of Adipogenesis regulatory factor. 
     In one embodiment, the method comprises determining the level of Ras-related protein Rab-2A. 
     In one embodiment, the method comprises determining the level of Ras-related protein Rab-3B. 
     In one embodiment, the method comprises determining the level of Ras-related protein Rab-7a. 
     In one embodiment, the method comprises determining the level of Protein DJ-1. 
     In one embodiment, the method comprises determining the level of Tetraspanin-6. 
     In one embodiment, the method comprises determining the level of Ras-related protein Rab-3D. 
     In one embodiment, the method comprises determining the level of Protein S100-P. 
     In one embodiment, the method comprises determining the level of Proton myo-inositol cotransporter. 
     In one embodiment, the method comprises determining the level of Plastin-2. 
     In one embodiment, the method comprises determining the level of Metalloreductase STEAP4. 
     In one embodiment, the method comprises determining the level of ADP-ribosylation factor-like protein 8B. 
     In one embodiment, the method comprises determining the level of Ras-related protein Rab-6A. 
     In one embodiment, the method comprises determining the level of Vesicle-associated membrane protein 2. 
     In one embodiment, the method comprises determining the level of Prenylcysteine oxidase 1. 
     In one embodiment, the method comprises determining the level of Sorcin. 
     In one embodiment, the method comprises determining the level of Grancalcin. 
     In one embodiment, the method comprises determining the level of Flotillin-1. 
     In one embodiment, the method comprises determining the level of Flotillin-2. 
     In some embodiments, the level of a single polypeptide (protein) is determined. In other embodiments, the level of more than one of the polypeptides is determined (e.g. the level of two or more polypeptides, or three or more polypeptides, or four or more polypeptides is determined). By “more than one” is meant 2, 3, 4, 5, 6, 7, 8, 9, 10 etc. . . . 246 (including all integers between 2 and 246). A determination of the level of each and every possible combination of the polypeptides can be performed. 
     Thus, in some embodiments multi-marker methods are performed. Determining the level of multiple of the polypeptides (biomarker multiplexing) may improve screening (e.g. diagnostic) accuracy. 
     In a preferred embodiment, the level of two of the stated polypeptides is determined. In another preferred embodiment, the level of three of the stated polypeptides is determined. In yet another preferred embodiment, the level of four of the stated polypeptides is determined. 
     In some embodiments, the level of a polypeptide selected from the group consisting of Transmembrane protein 256, Adipogenesis regulatory factor, Ragulator complex protein LAMTOR1, Ras-related protein Rab-2A, Ras-related protein Rab-3B, Ras-related protein Rab-3D, Ras-related protein Rab-7a, V-type proton ATPase 16 kDa proteolipid subunit, Metalloreductase STEAP4, Protein DJ-1, Protein S100-P, Synaptotagmin-like protein 4, Proton myo-inositol cotransporter and Tetraspanin-6 is determined in combination with determining the level of one of the other polypeptides set forth in Table 3 herein. Such a method is an example of a two-marker test. However, these combinations of two-markers can also be used in tests where a greater number of markers are determined. 
     In some embodiments, the level of a polypeptide selected from the group consisting of Plastin-2, ADP-ribosylation factor-like protein 8B and Ras-related protein Rab-6A is determined in combination with determining the level of two further of the stated polypeptides (e.g. two of the other polypeptides set forth in Table 3). Such a method is an example of a three-marker test. However, these combinations of three-markers can also be used in tests where a greater number of markers are determined. 
     Some preferred two-, three- and four-marker (polypeptide) combinations are described in Table 5 and are set out below:
         Transmembrane protein 256 and Ragulator complex protein LAMTOR1   Transmembrane protein 256 and V-type proton ATPase 16 kDa proteolipid subunit   Transmembrane protein 256 and Synaptotagmin-like protein 4   Transmembrane protein 256 and Claudin-3   Transmembrane protein 256 and Protein S100-A6   Transmembrane protein 256 and UDP-glucose 6-dehydrogenase   Adipogenesis regulatory factor and Ragulator complex protein LAMTOR1   Ragulator complex protein LAMTOR1 and Ras-related protein Rab-2A   Ragulator complex protein LAMTOR1 and Ras-related protein Rab-3B   Ragulator complex protein LAMTOR1 and Ras-related protein Rab-7a   Ragulator complex protein LAMTOR1 and Protein DJ-1   Ragulator complex protein LAMTOR1 and Synaptotagmin-like protein 4   Ragulator complex protein LAMTOR1 and Tetraspanin-6   V-type proton ATPase 16 kDa proteolipid subunit and Ras-related protein Rab-3B   V-type proton ATPase 16 kDa proteolipid subunit and Ras-related protein Rab-3D   V-type proton ATPase 16 kDa proteolipid subunit and Protein DJ-1   V-type proton ATPase 16 kDa proteolipid subunit and Protein S100-P   Synaptotagmin-like protein 4 and Proton myo-inositol cotransporter   Ragulator complex protein LAMTOR1 and Plastin-2 and Metalloreductase STEAP4   Ragulator complex protein LAMTOR1 and ADP-ribosylation factor-like protein 8B and Ras-related protein Rab-6A   Ragulator complex protein LAMTOR1 and Metalloreductase STEAP4 and Protein S100-P   Transmembrane protein 256 and Adipogenesis regulatory factor and   Ragulator complex protein LAMTOR1 and V-type proton ATPase 16 kDa proteolipid subunit       

     However, these combinations of two-, three- and four-markers can also be used in tests where a greater number of markers are determined. 
     Thus, in preferred methods of the present invention, the level of both of the polypeptides set forth above in the specific two marker combinations is determined. In other preferred methods the level of all three of the polypeptides set forth above in the specific three- marker combinations is determined. In another preferred method, the level of all four of the polypeptides set forth above in the specific four- marker combination is determined. 
     In another embodiment, the method comprises determining the level of Transmembrane protein 256 in combination with (i.e. and) determining the level of at least one (e.g. 1, 2 or 3) of the other polypeptides (proteins) set forth in Tables 1, 2 or 3 herein. In a particular embodiment, the method comprises determining the level of Transmembrane protein 256 in combination with (i.e. and) determining the level of at least one (e.g. 1, 2 or 3) of the other polypeptides (proteins) identified in Table 2 (or Table 3) herein as having a “sensitivity” of more than 60%. 
     Other appropriate combinations of markers can be derived from Table 3 by combining two or more of the markers in Table 3 (e.g. 2, 3, 4, 5 or 6 markers, preferably 2, 3 or 4 markers) that results in one or more of the patients (P) (preferably the majority of the patients, e.g. 9, 10, 11, 12, 13, 14, 15 or 16 of the patients, more preferably all of the patients) being associated with a positive call (as indicated by a “1” in Table 3) from at least one marker in the combination. Put another way, other appropriate combinations of markers (sequences/polypeptides) can be derived from Table 3 by combining two or more of the sequence rows (e.g. 2, 3, 4, 5 or 6 sequence rows, preferably 2, 3 or 4 sequence rows) such that the combination of said two or more sequence rows has at least one positive call (as indicated by “1”) in one or more patient columns (P) (preferably the majority of the patient columns, e.g. 9, 10, 11, 12, 13, 14, 15 or 16 of the patient columns, more preferably all of the patient columns). By way of example, sequences (markers) 12, 13 and 14 would be an appropriate three-marker combination as when sequence rows 12, 13 and 14 are combined there is at least one positive call (“1”) in all of the patient columns (P). 
     In some embodiments, the method comprises determining the level of one or more of the polypeptides (proteins) as set forth in Table 6 herein (e.g. Prenylcysteine oxidase 1) in combination with (“and”) determining the level of one or more of the other polypeptides mentioned herein (for example in combination with determining the level of one or more of Vesicle-associated membrane protein 2, Prenylcysteine oxidase 1, Sorcin or Grancalcin, or for example in combination with determining the level of one or more of the other polypeptides in Table 3, or for example in combination with determining the level of one or more of the other polypeptides in Table 6). 
     In some embodiments, the method comprises determining the level of one or more (1, 2, 3, 4, 5 or 6) of the polypeptides selected from the group consisting of: Transmembrane protein 256, Ragulator complex protein LAMTOR1, Ras-related protein Rab-3B, Flotillin-1, Flotillin-2 and Protein DJ-1 in combination with (“and”) determining the level of one or more of the other polypeptides mentioned herein (for example in combination with determining the level of one or more of the other polypeptides in Table 3, or for example in combination with determining the level of one or more of the other polypeptides in Table 6). 
     In some embodiments, the method comprises determining the level of one or more (1, 2, 3, 4 or 5) of the polypeptides selected from the group consisting of: Transmembrane protein 256, Ragulator complex protein LAMTOR1, Ras-related protein Rab-3B, Flotillin-1 and Flotillin-2 in combination with (“and”) determining the level of one or more of the other polypeptides mentioned herein (for example in combination with determining the level of one or more of the other polypeptides in Table 3, or for example in combination with determining the level of one or more of the other polypeptides in Table 6). 
     In some embodiments of the present invention the level of one or more (or all) of the following polypeptides (proteins) is not determined: 14-3-3 protein sigma, 14-3-3 protein theta, Actin-related protein ⅔ complex subunit 4, Actin-related protein ⅔ complex subunit 5, ADP-ribosylation factor-like protein 8B, Annexin A3, Beta-2-microglobulin, Calcium-binding protein 39, Calmodulin, CD81 antigen, CD9 antigen, Claudin-3, Destrin, Ferritin heavy chain, Flotillin-1, Myristoylated alanine-rich C-kinase substrate, Plastin-2, Protein DJ-1, Ras-related protein Rab-10, Ras-related protein Rab-12, Ras-related protein Rab-14, Ras-related protein Rab-1A, Ras-related protein Rab-1B, Ras-related protein Rab-7a, Ras-related protein Rab-8A, Ras-related protein Rab-8B, Septin-2, Translationally-controlled tumor protein, Vesicle-associated membrane protein 2. 
     In some embodiments of the present invention the level of one or more (or all) of the following polypeptides (proteins) is not determined: ADP-ribosylation factor-like protein 8B, Calmodulin, CD81 antigen, Claudin-3, Plastin-2, Protein DJ-1, Ras-related protein Rab-7a. 
     In some embodiments of the present invention the level of one or more (or all) of the following polypeptides (proteins) is not determined: Septin-2, CD81 antigen, Myristoylated alanine-rich C-kinase substrate, Ras-related protein Rab-14, Peptidyl-prolyl cis-trans isomerase FKBP1A. 
     In some embodiments of the present invention the level of transmembrane protease serine 2 is not determined. 
     In some embodiments of the present invention the level of prostate-specific antigen is not determined. 
     In some embodiments of the present invention the level of one or more (or all) of the following polypeptides (proteins) is not determined: Adipogenesis regulatory factor, Plastin-2, Ras-related protein Rab-2A, Ras-related protein Rab-3B, Ras-related protein Rab-3D, Metalloreductase STEAP4, Protein DJ-1, Protein S100-P, GDP-mannose 4.6 dehydratase, Lysosome membrane protein 2, 3-hydroxybutyrate dehydrogenase type 2, Protein S100-A6, 2′-deoxynucleoside 5′-phosphate N-hydrolase 1, Acid ceramidase, CD59 glycoprotein, CD81 antigen, Ragulator complex protein LAMTORS, Spermine synthase, Tumor protein D52, Zinc-alpha-2-glycoprotein, Alpha-actinin-1, Beta-2-microglobulin, Lipid phosphate phosphohydro lase 1, 14-3-3 protein sigma, Gamma-synuclein, Inter-alpha-trypsin inhibitor heavy chain H4, Aldehyde dehydrogenase family 1 member A3, Annexin A3, Cathepsin D, N(G).N(G)-dimethylarginine dimethylaminohydrolase 1, Proactivator polypeptide, Prostate-specific antigen, Protein Niban, Glutamate carboxypeptidase 2, Glutathione synthetase, Myristoylated alanine-rich C-kinase substrate, NAD(P)H-hydrate epimerase, Phosphoacetylglucosamine mutase, Sorcin, Adenine phosphoribosyltransferase, Costars family protein ABRACL, Lactotransferrin, Purine nucleoside phosphorylase, Voltage-dependent anion-selective channel protein 1, Collagen alpha-1(VI) chain, CD9 antigen, Flotillin-1, Mannose-1-phosphate guanyltransferase beta, Proteasome subunit alpha type-7, Pancreatic secretory granule membrane major glycoprotein GP2, Peptidyl-prolyl cis-trans isomerase FKBP1A, Flavin reductase (NADPH), Ras-related protein Rab-10, Heme-binding protein 2, Fatty acid-binding protein. epidermal, Proteasome subunit alpha type-5, Eukaryotic translation initiation factor 4H, Cellular retinoic acid-binding protein 2, L-xylulose reductase, Protein S100-A9, Alpha/beta hydrolase domain-containing protein 14B, Glutathione S-transferase P, Transmembrane protease serine 2, Ferritin heavy chain, Cathepsin Z, Annexin A4, Septin-2, Glutathione S-transferase Mu 3, Proteasome subunit beta type-2, Glutathione S-transferase Mu 1, Specifically androgen-regulated gene protein, ADP-ribosylation factor 5, Isocitrate dehydrogenase [NADP] cytoplasmic. 
     Exemplary amino acid sequences of the above named polypeptides are provided herein by reference to the corresponding Uniprot Accession Number (see e.g. Table 1 herein) (http://www.uniprot.org/). 
     As discussed above, the present invention provides a method for screening for prostate cancer in a subject. Alternatively viewed, the present invention provides a method of diagnosing prostate cancer in a subject. Alternatively viewed, the present invention provides a method for the prognosis of prostate cancer in a subject (prognosis of the future severity, course and/or outcome of prostate cancer). Alternatively viewed, the present invention provides a method of determining the clinical severity of prostate cancer in a subject. Alternatively viewed, the present invention provides a method for predicting the response of a subject to therapy. Alternatively viewed, the present invention provides a method for detecting the recurrence of prostate cancer. Alternatively viewed, the present invention provides a method of assessing qualification of a subject for a first (or follow-up) biopsy (prostate biopsy). Alternatively viewed, the present invention provides a method for determining the aggresiveness of prostate cancer, e.g. distinguishing between indolent and aggressive cancer (and thus may e.g. inform a decision between active surveillance and treatment). Alternatively viewed, the present invention provides a method of monitoring a subject (patient) under active surveillance. 
     Thus, the method of screening for prostate cancer in accordance with the present invention can be used, for example, for diagnosing prostate cancer, for the prognosis of prostate cancer, for monitoring the progression of prostate cancer, for determining the clinical severity of prostate cancer, for predicting the response of a subject to therapy, for determining the efficacy of a therapeutic regime being used to treat prostate cancer, for detecting the recurrence of prostate cancer, for assessing qualification of a subject for a first (or follow-up) biopsy (prostate biopsy), for distinguishing between indolent and aggressive cancer, or for monitoring a subject (patient) under active surveillance. 
     Thus, in one aspect the present invention provides a method for diagnosing prostate cancer in a subject. In some embodiments, a positive diagnosis is made if the level of one or more of the polypeptides (proteins/biomarkers) in the sample is altered (increased or decreased as the case may be) in comparison to a control level. Polypeptides for which an increased level is indicative of (e.g. diagnostic of) prostate cancer are described herein. Polypeptides for which a decreased level is indicative of (e.g. diagnostic of) prostate cancer are described herein. 
     In another aspect, the present invention provides a method for selecting patients suspected of having prostate cancer for further diagnosis, such as a first or a follow-up biopsy procedure. In some embodiments, a positive indication is made if the level of one or more of the polypeptides (proteins/biomarkers) in the sample is altered (increased or decreased as the case may be) in comparison to a control level. Polypeptides for which an increased level is indicative of (e.g. diagnostic of) prostate cancer are described herein. Polypeptides for which a decreased level is indicative of (e.g. diagnostic of) prostate cancer are described herein. 
     In another aspect, the present invention provides a method for determining whether a patient is likely to have an indolent or aggressive form of prostate cancer. In some embodiments, the prostate cancer is designated as aggressive if the level of one or more of the polypeptides (proteins/biomarkers) in the sample is altered (increased or decreased as the case may be) in comparison to a control level. 
     In another aspect, the present invention provides a method for the prognosis of prostate cancer in a subject. In such methods the level of one or more of polypeptides (proteins/biomarkers) discussed above in the sample is indicative of the future severity, course and/or outcome of prostate cancer. For example, an alteration (increase or decrease as the case may be) in the level of one or more of the polypeptides (proteins/biomarkers) in the sample in comparison to a control level may indicate a poor prognosis. A highly altered level may indicate a particularly poor prognosis. 
     Thus, in some embodiments, an increased level of one or more of the polypeptides for which an increased level is indicative of prostate cancer is suggestive of (i.e. indicative of) a poor prognosis. In some embodiments, a decreased level of one or more of the polypeptides for which a decreased level is indicative of prostate cancer is suggestive of (i.e. indicative of) a poor prognosis. Conversely, if one or more polypeptides has an unaltered level (or an essentially unaltered level) that can be indicative of a good prognosis. 
     Serial (periodic) measuring of the level of one or more of the polypeptides (proteins/biomarkers) may also be used for prognostic purposes looking for either increasing or decreasing levels over time. In some embodiments, an altering level (increase or decrease) of one or more of the polypeptides over time (in comparison to a control level) may indicate a worsening prognosis. In some embodiments, an altering level (increase or decrease) of one or more of the polypeptides over time (in comparison to a control level) may indicate an improving prognosis. Thus, the methods of the present invention can be used to monitor disease progression. Such monitoring can take place before, during or after treatment of prostate cancer by surgery or therapy. Thus, in one aspect the present invention provides a method for monitoring the progression of prostate cancer in a subject. 
     Methods of the present invention can be used in the active monitoring of patients which have not been subjected to surgery or therapy, e.g. to monitor the progress of prostate cancer in untreated patients. Again, serial measurements can allow an assessment of whether or not, or the extent to which, the prostate cancer is worsening, thus, for example, allowing a more reasoned decision to be made as to whether therapeutic intervention is necessary or advisable. 
     Monitoring can also be carried out, for example, in an individual who is thought to be at risk of developing prostate cancer, in order to obtain an early, and ideally pre-clinical, indication of prostate cancer. 
     In another aspect, the present invention provides a method for determining the clinical severity of prostate cancer in a subject. In such methods the level of one or more of the polypeptides (proteins/biomarkers) in the sample shows an association with the severity of the prostate cancer. Thus, the level of one or more of polypeptides is indicative of the severity of the prostate cancer In some embodiments, the more altered (more increased or more decreased as the case may be) the level of one or more of the polypeptides in comparison to a control level, the greater the likelihood of a more severe form of prostate cancer. In some embodiments the methods of the invention can thus be used in the selection of patients for therapy. 
     Serial (periodical) measuring of the level of one or more of the polypeptides (proteins/biomarkers) may also be used to monitor the severity of prostate cancer looking for either increasing or decreasing levels over time. Observation of altered levels (increase or decrease as the case may be) may also be used to guide and monitor therapy, both in the setting of subclinical disease, i.e. in the situation of “watchful waiting” (also known as “active surveillance”) before treatment or surgery, e.g. before initiation of pharmaceutical therapy, or during or after treatment to evaluate the effect of treatment and look for signs of therapy failure. 
     The present invention also provides a method for predicting the response of a subject to therapy. In such methods the choice of therapy may be guided by knowledge of the level of one or more of the polypeptides in the sample. 
     The present invention also provides a method of determining (or monitoring) the efficacy of a therapeutic regime being used to treat prostate cancer. In such methods, an alteration (increase or decrease as the case may be) in the level of one or more of the polypeptides indicates the efficacy of the therapeutic regime being used. For example, if the level of one or more of the polypeptides for which an increased level is indicative of prostate cancer is reduced during (or after) therapy, this is indicative of an effective therapeutic regime. Conversely, for example, if the level of one or more of the polypeptides for which a decreased level is indicative of prostate cancer is increased during (or after) therapy, this is indicative of an effective therapeutic regime. In such methods, serial (periodical) measuring of the level of one or more of the polypeptides (proteins/biomarkers) over time can also be used to determine the efficacy of a therapeutic regime being used. 
     The present invention also provides a method for detecting the recurrence of prostate cancer. 
     The features and discussion herein in relation to the method of screening for prostate cancer (e.g. in relation to preferred polypeptides or combinations thereof discussed above) apply, mutatis mutandis, to the other related methods of present invention (e.g. to a method of diagnosing prostate cancer). 
     In one embodiment, the invention provides the use of the methods (e.g. screening, diagnostic or prognostic methods) in conjunction other known screening, diagnostic or prognostic methods (e.g. the PSA test). Thus, for example, the methods of the invention can be used to confirm a diagnosis of prostate cancer in a subject. In some embodiments the methods of the present invention are used alone. 
     A yet further aspect provides a kit for the screening (e.g. diagnosis or prognosis) of prostate cancer which comprises an agent suitable for determining the level of one or more of the polypeptides (proteins/biomarkers) described above, or fragments thereof, in a sample. Preferred agents are antibodies. In preferred aspects said kits are for use in the methods of the invention as described herein. Preferably, said kits comprise instructions for use of the kit components, for example in diagnosis. In some embodiments, the kit is a multimarker kit. Thus, in some embodiments the kit comprises more than one agent (e.g. two, three or four distinct agents), each agent being suitable for determining the level of one of the polypeptides (proteins/biomarkers) described above, or fragments thereof, in a sample. Using such kits (multimarker kits) the level of multiple (e.g. two, three or four) polypeptides may be determined. Exemplary groups (combinations) of polypeptides (markers) whose level may be determined using such multimarker kits are discussed elsewhere herein in relation to other aspects of the invention. In a preferred embodiment of such multimarker kits, the agent suitable for determining the level of a polypeptide is an antibody. 
     The level of the polypeptide (protein) in question can be determined by analysing the sample which has been obtained from or removed from the subject by an appropriate means. The determination is typically carried out in vitro. 
     Levels of one or more of the polypeptides in the sample can be measured (determined) by any appropriate assay, a number of which are well known and documented in the art and some of which are commercially available. The level of one or more of the polypeptides (proteins/biomarkers) can be determined e.g. by an immunoassay such as a radioimmunoassay (RIA) or fluorescence immunoassay, immunoprecipitation and immunoblotting (e.g. Western blotting) or Enzyme-Linked ImmunoSorbent Assay (ELISA). Immunoassays are a preferred technique for determining the levels of one or more of the polypeptides in accordance with the present invention. 
     Preferred assays are ELISA-based assays, although RIA-based assays can also be used effectively. Both ELISA- and RIA-based methods can be carried out by methods which are standard in the art and would be well known to a skilled person. Such methods generally involve the use of an antibody to a relevant polypeptide under investigation, or fragment thereof, which is incubated with the sample to allow detection of said polypeptide (or fragment thereof) in the sample. Any appropriate antibodies can be used and examples of these are described in the prior art. For example, an appropriate antibody to a polypeptide under investigation, or an antibody which recognises particular epitopes of said polypeptide, can be prepared by standard techniques, e.g. by immunization of experimental animals, which are know to a person skilled in the art. The same antibody to a given polypeptide under investigation or fragments thereof can generally be used to detect said polypeptide in either a RIA-based assay or an ELISA-based assay, with the appropriate modifications made to the antibody in terms of labelling etc., e.g. in an ELISA assay the antibodies would generally be linked to an enzyme to enable detection. Any appropriate form of assay can be used, for example the assay may be a sandwich type assay or a competitive assay. 
     In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be determined through the magnitude of the fluorescence. For RIA, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine attached to tyrosine. This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two chemically bind to one another. Then, a sample from a patient containing an unknown quantity of that same antigen is added. This causes the unlabeled (or “cold”) antigen from the sample to compete with the radiolabeled antigen for antibody binding sites. As the concentration of “cold” antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured. A binding curve can then be plotted, and the exact amount of antigen in the patient&#39;s sample can be determined. Measurements are usually also carried out on standard samples with known concentrations of marker (antigen) for comparison. 
     In some embodiments, the level of Flotillin-2 is determined by an ELISA-based assay. 
     In some embodiments, the level of Protein DJ-1 is determined by an ELISA-based assay. 
     In some embodiments, immunohistochemistry with appropriate antibodies could be carried out. 
     The use of immunoblotting (e.g. Western blotting) can also be used for measuring the level of one or more of the polypeptides in accordance with the present invention. 
     Preferred agents for use in determining the level of one or more of the polypeptides in accordance with the present invention are antibodies (antibodies to the polypeptide whose level is to be determined). 
     In other preferred embodiments, the level of one or more of the polypeptides in the sample can be measured (determined) by mass spectrometry. Suitable mass spectrometry methods (and associated data processing techniques) are well known and documented in the art. A particularly preferred mass spectrometry method (and associated data processing techniques) for determining the level of one or more of the polypeptides in the sample is described herein in the Example. In some embodiments mass spectrometry (and associated data processing techniques) is used to obtain a ratio of the level of a polypeptide in the sample in comparison to a control. 
     In accordance with the present invention, a quantitative, semi-quantitative or qualitative assessment (determination) of the level of one or more of the polypeptides can be made. 
     It is well understood in the art that when detecting the presence of a protein in a sample, it is not necessary to detect the presence of the full-length protein (i.e. the entire protein sequence); detecting the presence of a fragment of a protein can be indicative of the presence of the entire protein. 
     Thus, in certain embodiments of the methods of the invention described herein, any fragments of the polypeptides, in particular naturally occurring fragments, can be analysed as an alternative to the polypeptides themselves (full length polypeptides). Suitable fragments for analysis should be characteristic of the full-length protein. Suitable fragments can be at least 6 consecutive amino acids in length. For example, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200 or at least 500 consecutive amino acids in length. Suitable fragments can represent at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the length of the full-length polypeptide (protein). 
     In some embodiments the level of the full-length polypeptide is determined. 
     Reference herein to the “polypeptides” whose level is to be determined in accordance with the invention includes reference to all forms of said polypeptides (as appropriate) which might be present in a subject, including derivatives, mutants and analogs thereof, in particular fragments thereof or modified forms of the polypeptides or their fragments. Exemplary and preferred modified forms include forms of these molecules which have been subjected to post translational modifications such as glycosylation or phosphorylation. In some embodiments, the level of unmodified forms of the polypeptides (or their fragments) is determined. 
     The “increase” in the level or “increased” level of one or more of the polypeptides as described herein includes any measurable increase or elevation of the polypeptide (protein/biomarker) in question when the polypeptide in question is compared with a control level. Preferably, the level is significantly increased, compared to the level found in an appropriate control sample or subject. More preferably, the significantly increased levels are statistically significant, preferably with a probability value of &lt;0.05. Viewed alternatively, an increase in level of the polypeptide of ≧2%, ≧3%, ≧5%, ≧10%, ≧25%, ≧50%, ≧75%, ≧100%, ≧200%, ≧300%, ≧400%, ≧500%, ≧600%, ≧700%, ≧800%, ≧900%, ≧1000%, ≧2000%, ≧5000%, or ≧10,000% compared to the level found in an appropriate control sample or subject (i.e. when compared to a control level) is indicative of the presence of prostate cancer. In a preferred embodiment the increase is ≧75% compared to the level found in an appropriate control sample or subject. 
     In some embodiments, the increase in level (e.g. of Transmembrane protein 256, Ragulator complex protein LAMTOR1, Ras-related protein Rab-3B, Flotillin-1 or Flotillin-2) is ≧50%, ≧75%, ≧100%, ≧150%, ≧200%, ≧250% or ≧500% compared to the level found in an appropriate control sample or subject, for example as determined by Western blotting. 
     In some embodiments, the increase in level (e.g. of Flotillin-2 or Protein DJ-1) is ≧50%, ≧75%, ≧100%, ≧150%, ≧200%, ≧250% or ≧500% compared to the level found in an appropriate control sample or subject, for example as determined by an ELISA-based assay. 
     In some embodiments, for those polypeptides described herein whose level is increased in prostate cancer samples in comparison to a control level, a level (concentration) of at least 10 pg polypeptide/μg (total) exosomal protein, at least 25 pg polypeptide/μg (total) exosomal protein, at least 50 pg polypeptide/μg (total) exosomal protein, at least 100 pg polypeptide/μg (total) exosomal protein, at least 200 pg polypeptide/μg (total) exosomal protein, at least 300 pg polypeptide/μg (total) exosomal protein, at least 400 pg polypeptide/μg (total) exosomal protein, at least 0.5 ng polypeptide/μg (total) exosomal protein, at least 0.75 ng polypeptide/μg (total) exosomal protein, at least 1 ng polypeptide/μg (total) exosomal protein, at least 1.5 ng polypeptide/μg (total) exosomal protein, at least 2 ng polypeptide/μg (total) exosomal protein, at least 3 ng polypeptide/μg (total) exosomal protein, at least 4 ng polypeptide/μg (total) exosomal protein, at least 5 ng polypeptide/μg (total) exosomal protein, at least 10 ng polypeptide/μg (total) exosomal protein, at least 25 ng polypeptide/μg (total) exosomal protein, at least 50 ng polypeptide/μg (total) exosomal protein or at least 100 ng polypeptide/μg (total) exosomal protein in a sample is indicative of prostate cancer in a subject. In some embodiments, concentrations may be determined by an ELISA-based assay. 
     The “decrease” in the level or “decreased” level of one or more of the polypeptides as described herein includes any measurable decrease or reduction of the polypeptide (protein/biomarker) in question when the polypeptide in question is compared with a control level. Preferably, the level is significantly decreased, compared to the level found in an appropriate control sample or subject. More preferably, the significantly decreased levels are statistically significant, preferably with a probability value of &lt;0.05. Viewed alternatively, a decrease in level of the polypeptide of ≧2%, ≧3%, ≧5%, ≧10%, ≧25%, ≧50%, ≧75%, ≧100%, ≧200%, ≧300%, ≧400%, ≧500%, ≧600%, ≧700%, ≧800%, ≧900%, ≧1000%, ≧2000%, ≧5000%, or ≧10,000% compared to the level found in an appropriate control sample or subject (i.e. when compared to a control level) is indicative of the presence of prostate cancer. In a preferred embodiment the decrease is ≧50% compared to the level found in an appropriate control sample or subject. 
     In some embodiments, for those polypeptides described herein whose level is decreased in prostate cancer samples in comparison to a control level, a level (concentration) of less than 10 pg polypeptide/μg (total) exosomal protein, less than 25 pg polypeptide/μg (total) exosomal protein, less than 50 pg polypeptide/μg (total) exosomal protein, less than 100 pg polypeptide/μg (total) exosomal protein, less than 200 pg polypeptide/μg (total) exosomal protein, less than 300 pg polypeptide/μg (total) exosomal protein, less than 400 pg polypeptide/μg (total) exosomal protein, less than 0.5 ng polypeptide/μg (total) exosomal protein, less than 0.75 ng polypeptide/μg (total) exosomal protein, less than 1 ng polypeptide/μg (total) exosomal protein, less than 1.5 ng polypeptide/μg (total) exosomal protein, less than 2 ng polypeptide/μg (total) exosomal protein, less than 3 ng polypeptide/μg (total) exosomal protein, less than 4 ng polypeptide/μg (total) exosomal protein, less than 5 ng polypeptide/μg (total) exosomal protein, less than 10 ng polypeptide/μg (total) exosomal protein, less than 25 ng polypeptide/μg (total) exosomal protein, less than 50 ng polypeptide/μg (total) exosomal protein or less than 100 ng polypeptide/μg (total) exosomal protein in a sample is indicative of prostate cancer in a subject. In some embodiments, concentrations may be determined by an ELISA-based assay. 
     A “control level” is the level of a polypeptide in a control subject (e.g. in a sample that has been obtained from a control subject). Appropriate control subjects or samples for use in the methods of the invention would be readily identified by a person skilled in the art. Such subjects might also be referred to as “normal” subjects or as a reference population. Examples of appropriate control subjects would include healthy subjects, for example, individuals who have no history of any form of prostate disease (e.g. prostate cancer) and no other concurrent disease, or subjects who are not suffering from, and preferably have no history of suffering from, any form of prostate disease, in particular individuals who are not suffering from, and preferably have no history of suffering from, prostate cancer. Preferably control subjects are not regular users of any medication. In a preferred embodiment control subjects are healthy subjects. 
     The control level may correspond to the level of the equivalent polypeptide in appropriate control subjects or samples, e.g. may correspond to a cut-off level or range found in a control or reference population. Alternatively, said control level may correspond to the level of the marker (polypeptide) in question in the same individual subject, or a sample from said subject, measured at an earlier time point (e.g. comparison with a “baseline” level in that subject). This type of control level (i.e. a control level from an individual subject) is particularly useful for embodiments of the invention where serial or periodic measurements of polypeptide levels in individuals, either healthy or ill, are taken looking for changes in the levels of the polypeptide(s). In this regard, an appropriate control level will be the individual&#39;s own baseline, stable, nil, previous or dry value (as appropriate) as opposed to a control or cutoff level found in the general population. Control levels may also be referred to as “normal” levels or “reference” levels. The control level may be a discrete figure or a range. 
     Although the control level for comparison could be derived by testing an appropriate set of control subjects, the methods of the invention would not necessarily involve carrying out active tests on control subjects as part of the methods of the present invention but would generally involve a comparison with a control level which had been determined previously from control subjects and was known to the person carrying out the methods of the invention. 
     The sample which is tested according to the methods of the invention is a sample comprising urinary exosomes. In accordance with the present invention urinary exosomes can comprise (contain), or be suspected of comprising (containing), the polypeptide(s) (exosomal polypeptides/ exosomal proteins) whose level is to be determined. In other words, the methods of the invention involve the determination of levels of one or more polypeptides that are present in urinary exosomes (exosomes present in the urine). Exosomes are typically 30-150nm vesicles released by cells. Typically the sample has been obtained from (removed from) a subject, preferably a human male subject. In other aspects, the method further comprises a step of obtaining a sample from the subject. 
     In some embodiments the sample is a urine sample. In some embodiments the sample is derived from urine. Urine (and samples derived from urine e.g. isolated or partially isolated urinary exosomes) represents an attractive type of sample because it is easy to obtain (non-invasively) and its composition can reflect changes in the functioning of the prostate and other organs of the urogenital tract. In addition, the composition of urine is less complex than the composition of some other sample types, e.g. blood. In some embodiments the urine sample is used (processed) within 2 hours of having being collected from the subject. In some embodiments the urine sample is collected in the morning. In some embodiments, the urine sample may be a urine sample that has been collected without performing prostatic massage prior to urine collection. In some embodiments, the sample may be a sample derived from urine (e.g. isolated or partially isolated urinary exosomes), wherein said urine has been collected without performing prostatic massage prior to urine collection. 
     The term “sample” also encompasses any material derived by processing a biological sample (e.g. derived by processing a urine sample). Derived materials include isolated (or substantially or partially isolated) urinary exosomes from the sample. Processing of biological samples to obtain a test sample may involve one or more of: filtration, distillation, centrifugation, extraction, concentration, dilution, purification, inactivation of interfering components, addition of reagents, and the like. In some methods of the present invention, a sample comprising urinary exosomes (e.g. a urine sample) is subjected to a processing step, e.g. to isolate or partially isolate urinary exosomes, e.g. as described elsewhere herein. 
     In a preferred embodiment the sample comprises (or consists of or consists essentially of) isolated urinary exosomes. By isolated urinary exosomes is meant that the urinary exosomes are free from (or substantially free from) other urine components. Thus, in a preferred embodiment the sample is an isolated (or purified) sample of urinary exosomes. Isolated (e.g. purified) urinary exosomes can be resuspended in (or mixed with) an appropriate buffer (e.g. PBS) prior to analysis. Samples can contain urinary exosomes (e.g. isolated or purified urinary exosomes) and other non-urine components. 
     Any suitable method for isolating urinary exosomes may be employed. Urinary exosomes may be isolated from urine by serial centrifugation. A suitable method for isolating urinary exosomes by serial centrifugation is described herein in the Example. In this exemplary method, urine is centrifuged at 2,000 g for 15 min, and then at 10,000 g for 30 min discarding the pellet at each step. The exosomes present in the supernatant are then pelleted at 100,000 g for 70 min and washed with PBS. Exosomes are then resuspended again in PBS, filtrated through a 200 nm pore filter and pelleted at 100,000 g for 70 min. The pellet is resuspended in 50-100 μ1PBS and stored at -80 ° C. Thus, urinary exosomes for use in the methods of the present invention are capable of being isolated by such a serial centrifugation method. 
     Another suitable method for isolating urinary exosomes is to use antibody capture with an antibody that specifically binds to exosomal membrane proteins. Moreover, to specifically isolate urinary exosomes that originate from prostate cells, an antibody against a prostate-specific protein could be used. Antibodies can be bound to a bead or particle that facilitates isolation of urinary exosomes. 
     Commercially available kits may be used for the isolation of exosomes. Such kits include, but are not limited to, kits from Life Technologies (Catalogue number #4484452), Exiqon (Catalogue number #300102), Norgen Biotek Corp (Catalogue number #47200), System Biosciences (Catalogue number #EXOTC 50A-1), Cell Guidance Systems (Catalogue number #EX01) and 101 Bio (Catalogue number #P120). 
     In some embodiments, urinary exosomes are enzymatically (e.g. trypsin) digested (e.g. in solution digestion) prior to analysis of the levels of polypeptides therein. Such enzymatic digestion of urinary exosomes is typically performed when the level of one or more of the polypeptides therein is to be determined using mass spectrometry. An appropriate protocol for the enzymatic digestion of urinary exosomes prior to mass spectrometry analysis is provided herein in the Example. 
     In some embodiments, the urinary exosomes are disrupted (e.g. denatured) prior to determination of the level of one or more of the polypeptides therein. 
     Samples can be used immediately or can be stored for later use (e.g. at −80° C.). 
     In some embodiments, relatively low amounts of urinary exosomes are required in order to detect (e.g. by Western blot) a polypeptide whose level is to be determined. Thus, in some embodiments, the sample may comprise less than 10 μg exosomal protein, less than 5 μg exosomal protein, less than 2 μg exosomal protein, less than 1 iug exosomal protein, less than 0.5 μg exosomal protein, less than 0.25 μg exosomal protein, less than 100 ng exosomal protein, less than 50 ng exosomal protein or less than 25 ng exosomal protein. In some embodiments, the sample may comprise at least 25 ng exosomal protein, at least 50 ng exosomal protein, at least 100 ng exosomal protein, at least 0.25 μg exosomal protein, at least 0.5 μg exosomal protein, at least 1 μg exosomal protein, at least 2 μg exosomal protein, at least 5 μg exosomal protein or at least 10 μg exosomal protein. Exosomal protein may be total exosomal protein. 
     The methods of the invention as described herein can be carried out on any type of subject which is capable of suffering from prostate cancer. The methods are generally carried out on mammals, for example humans, primates (e.g. monkeys), laboratory mammals (e.g. mice, rats, rabbits, guinea pigs), livestock mammals (e.g. horses, cattle, sheep, pigs) or domestic pets (e.g. cats, dogs). Preferably the subject is a human. 
     In one embodiment, the subject (e.g. a human) is a subject at risk of developing prostate cancer or at risk of the occurrence of prostate cancer (e.g. a healthy subject or a subject not displaying any symptoms of prostate cancer or any other appropriate “at risk” subject). In another embodiment the subject is a subject having, or suspected of having (or developing), prostate cancer. 
     In some aspects, a method of the invention may further comprise an initial step of selecting a subject (e.g. a human subject) at risk of developing prostate cancer or having, or suspected of having (or developing), prostate cancer. The subsequent method steps can be performed on a sample from such a selected subject. 
     In another aspect, the present invention provides method of screening for prostate cancer in a subject, said method comprising 
     determining the level in a sample of one or more polypeptides selected from the group consisting of:
         Transmembrane protein 256, Adipogenesis regulatory factor, Ragulator complex protein LAMTOR1, Plastin-2, Ras-related protein Rab-2A, Ras-related protein Rab-3B, Ras-related protein Rab-3D, Ras-related protein Rab-7a, V-type proton ATPase 16 kDa proteolipid subunit, Metalloreductase STEAP4, Protein DJ-1, Protein S100-P, Synaptotagmin-like protein 4, ADP-ribosylation factor-like protein 8B, Proton myo-inositol cotransporter, Ras-related protein Rab-6A, Tetraspanin-6, Claudin-10, Claudin-2, Claudin-3, GDP-mannose 4.6 dehydratase, Glucosamine-6-phosphate isomerase 1, Lysosome membrane protein 2, Major facilitator superfamily domain-containing protein 12, Melanophilin, Sepiapterin reductase, Thioredoxin domain-containing protein 17, 3-hydroxybutyrate dehydrogenase type 2, Calmodulin, Carboxypeptidase Q, Flotillin-2, Galectin-3-binding protein, P2X purinoceptor 4, Protein dopey-2, Protein S100-A6, Ras-related protein Rab-35, Serine/threonine-protein phosphatase 2A catalytic subunit alpha iso form, 2′-deoxynucleoside 5′-phosphate N-hydrolase 1, Acid ceramidase, Calbindin, CD59 glycoprotein, CD81 antigen, Cytochrome b561, Enolase-phosphatase E1, Golgi phosphoprotein 3, Nicastrin, Probable serine carboxypeptidase CPVL, Ragulator complex protein LAMTORS, Ras-related protein Rab-27B, Secretory carrier-associated membrane protein 2, Spermine synthase, S-phase kinase-associated protein 1, Transmembrane 7 superfamily member 3, Tumor protein D52, Ubiquitin-conjugating enzyme E2 variant 2, UDP-glucose 6-dehydrogenase, Zinc-alpha-2-glycoprotein, Glycerophosphodiester phosphodiesterase domain-containing protein 3, EF-hand domain-containing protein D2, Ras-related protein Rab-14, Omega-amidase NIT2, Alpha-actinin-1, Monocarboxylate transporter 5, Ras-related protein Rab-12, Ras-related protein Rab-8A, Transmembrane protein 63A, Beta-2-microglobulin, V-type proton ATPase subunit d 1, Lipid phosphate phosphohydrolase 1, Integral membrane protein GPR155, 14-3-3 protein sigma, E3 ubiquitin-protein ligase LRSAM1, HLA class II histocompatibility antigen. DM alpha chain, Ras-related protein Rab-9A, Src substrate cortactin, Aquaporin-7, Gamma-synuclein, 14-3-3 protein theta, Aspartate aminotransferase. cytoplasmic, Chloride intracellular channel protein 3, Destrin, GTPase HRas, Prostaglandin reductase 2, T-complex protein 1 subunit epsilon, Inter-alpha-trypsin inhibitor heavy chain H4, Aldehyde dehydrogenase family 1 member A3, Annexin A3, Battenin, Cathepsin D, N(G).N(G)-dimethylarginine dimethylaminohydrolase 1, Neural proliferation differentiation and control protein 1, Proactivator polypeptide, Prostate-specific antigen, Protein lifeguard 3, Protein Niban, Protein spinster homolog 1, Ragulator complex protein LAMTOR2, Ragulator complex protein LAMTOR3, Synaptotagmin-7, Transmembrane protein 106B, Unconventional myosin-Vc, Vesicle-associated membrane protein 2, V-type proton ATPase subunit F, A-kinase anchor protein 7 isoforms alpha and beta, Arylsulfatase F, C-Jun-amino-terminal kinase-interacting protein 4, Deleted in malignant brain tumors 1 protein, Glia maturation factor beta, Glutamate carboxypeptidase 2, Glutathione synthetase, Hippocalcin-like protein 1, Myristoylated alanine-rich C-kinase substrate, NAD(P)H-hydrate epimerase, Napsin-A, Phosphoacetylglucosamine mutase, Probable phospholipid-transporting ATPase IA, Quinone oxidoreductase, Retinol-binding protein 5, Serine incorporator 2, Solute carrier family 15 member 1, Sorcin, Sucrase-isomaltase. Intestinal, Actin-related protein ⅔ complex subunit 4, Actin-related protein ⅔ complex subunit 5, Adenine phosphoribosyltransferase, Costars family protein ABRACL, Ig alpha-1 chain C region, Interferon-induced transmembrane protein 3, Lactotransferrin, Purine nucleoside phosphorylase, Syntaxin-binding protein 4, Tetraspanin-8, Vacuolar protein-sorting-associated protein 25, Voltage-dependent anion-selective channel protein 1, Collagen alpha-1(VI) chain, Signal transducing adapter molecule 1, CD9 antigen, Flotillin-1, Grancalcin, Mannose-1-phosphate guanyltransferase beta, Proteasome subunit alpha type-7, Ras-related protein Rab-18, Vacuolar protein sorting-associated protein 37C, Signal transducing adapter molecule 2, Pancreatic secretory granule membrane major glycoprotein GP2, 1-phosphatidylinosito14.5-bisphosphate phosphodiesterase gamma-2, Lysosome-associated membrane glycoprotein 2, Cysteine-rich C-terminal protein 1, Plasma serine protease inhibitor, Tyrosine-protein kinase Lck, Ragulator complex protein LAMTOR4, Secretory carrier-associated membrane protein 1, Peptidyl-prolyl cis-trans isomerase FKBP1A, Transmembrane protein 176A, Thymosin beta-4, Haloacid dehalogenase-like hydrolase domain-containing protein 2, Cell division control protein 42 homolog, Ras-related protein Rab-17, Chloride intracellular channel protein 6, Choline transporter-like protein 4, Flavin reductase (NADPH), Ras-related protein Rab-10, Heme-binding protein 2, Fatty acid-binding protein. epidermal, Small integral membrane protein 5, Lysosomal-associated transmembrane protein 4A, Phytanoyl-CoA dioxygenase domain-containing protein 1, Proteasome subunit alpha type-5, Calmodulin-like protein 3, Presenilin-1, Ribosyldihydronicotinamide dehydrogenase [quinone], Translationally-controlled tumor protein, Lysosome-associated membrane glycoprotein 1, ADP-ribosyl cyclase 1, Myotrophin, Dynein light chain 2. cytoplasmic, Dihydropteridine reductase, Nicotinate-nucleotide pyrophosphorylase [carboxylating], Cell cycle control protein 50A, Phosphomevalonate kinase, Eukaryotic translation initiation factor 4H, Protein tweety homo log 3, Sodium-dependent phosphate transport protein 2B, Ig lambda-2 chain C regions, Cellular retinoic acid-binding protein 2, Protein CutA, Proteasome subunit alpha type-4, Solute carrier family 35 member F6, Delta-aminolevulinic acid dehydratase, L-xylulose reductase, Uroplakin-1a, Cornifin-A, Zinc finger protein 185, Transmembrane protein 8A, Prenylcysteine oxidase 1, Lysozyme C, Paralemmin-1, Carcinoembryonic antigen-related cell adhesion molecule 6, Sodium/glucose cotransporter 1, Prostaglandin reductase 1, Protein S100-A9, MICAL-like protein 1, Alpha/beta hydrolase domain-containing protein 14B, Aquaporin-2, Glutathione S-transferase P, Probable almitoyltransferase ZDHHC1, Ras-related protein Rab-8B, Transmembrane protease serine 2, Ras-related protein Rab-1B, Ras-related protein Rab-1A, Ras-related protein Rab-43, Synaptogyrin-2, HLA class II histocompatibility antigen. DRB1-15 beta chain, Lysosomal protein NCU-G1, Calcium-binding protein 39, Dynamin-2, CDC42 small effector protein 2, Ferritin heavy chain, Solute carrier family 35 member F2,Probable hydrolase PNKD, Cathepsin Z, Tubulin beta-2B chain, Thiosulfate sulfurtransferase/rhodanese-like domain-containing protein 1, Mitogen-activated protein kinase 1, Alcohol dehydrogenase class-3, Low molecular weight phosphotyrosine protein phosphatase, Annexin A4, Septin-2, Glutathione S-transferase Mu 3, Protein BRICK1, Proteasome subunit beta type-2, Ubiquitin- conjugating enzyme E2 K, Protein S100-A1, Microtubule-associated protein 1A, Glutathione S-transferase Mu 1, Matrix metalloproteinase-24, Small integral membrane protein 22, Heparan-alpha-glucosaminide N-acetyltransferase, Specifically androgen-regulated gene protein, Abl interactor 1, Uncharacterized protein C6orf132, ADP-ribosylation factor 5, Isocitrate dehydrogenase [NADP] cytoplasmic, Glycolipid transfer protein, Tropomyosin alpha-4 chain and Natural resistance-associated macrophage protein 2;   wherein said sample comprises urinary exosomes and wherein said sample has been obtained from said subject; and   wherein an altered level in said sample of one or more of said polypeptides in comparison to a control level is indicative of prostate cancer in said subject.       

     The features and discussion herein in relation to other aspects of the invention (e.g. in relation to preferred polypeptides or combinations thereof discussed above) apply,  mutatis mutandis , to this aspect of the invention. 
     An altered level of one or more of the polypeptides as described herein includes any measurable alteration or change of the polypeptide (protein/biomarker) in question when the polypeptide in question is compared with a control level. An altered level includes an increased or decreased level. Preferably, the level is significantly altered, compared to the level found in an appropriate control sample or subject. More preferably, the significantly altered levels are statistically significant, preferably with a probability value of &lt;0.05. Exemplary altered levels are discussed above in relation to “increased” and “decreased” levels. 
     In some aspects, methods of the invention are provided which further comprise a step of treating prostate cancer by therapy (e.g. pharmaceutical therapy) or surgery (e.g. prostatectomy). For example, if the result of a method of the invention is indicative of the prostate cancer in the subject (e.g. a postive diagnosis of prostate cancer is made), then an additional step of treating prostate cancer by therapy or surgery can be performed. Methods of treating prostate cancer by therapy or surgery are known in the art. 
    
    
     
       The invention will be further described with reference to the following non-limiting Example with reference to the following drawings in which: 
         FIG. 1 : Scatterplots, displaying the range of values observed in the PAT and CTR samples, are shown for illustrative purposes for the three biomarkers with the highest individual sensitivities at the chosen threshold ( FIG. 1 ). For each sample type, PAT and CTR, the intensity (TOP3TIC) was plotted displaying the distribution along the y-axis. The iBAQ ratio and the number of samples (PAT and CTR) in which the protein was detected are also shown. LFQ: Label Free Quantification. 
         FIG. 2 : Scatterplots, displaying the range of values observed in the PAT and CTR samples for Vesicle-associated membrane protein 2, Prenylcysteine oxidase 1, Sorcin and Grancalcin, are shown. 
         FIG. 3 : Amount of exosomal protein required to detect specific biomarkers by Western blot. A. Different amounts of exosmal proteins were loaded on gels and specific proteins were detected by Western blot. B. Example where the quantity of the protein band (flotillin2 used as an example) is related to the amount of loaded urinary exosomes. FLOT2: flotillin2. 
         FIG. 4 : Patient (prostate cancer patient) to control ratios for the different proteins (biomarkers) based on Western blot detection of specific biomarkers in urinary exosomes of healthy males and prostate cancer patients. 
         FIG. 5 : Analysis of the diagnostic properties of flotillin2 based on Western blot detection. FLOT2: flotillin2. 
     
    
    
     EXAMPLES 
     Example 1 
     Results 
     Urinary exosomes from 15 healthy controls (CTR, C) and 17 prostate cancer patients (PAT, P) were isolated by serial centrifugation. In order to find exosomal proteins differently expressed in control versus prostate cancer patients, urinary exosomes were in-solution digested and analyzed using nanocapillary liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). This approach (termed “discovery analysis”) identified on average 1090 proteins per sample with 1% FDR. One patient sample, P11, was excluded from further analysis based on a much lower level of detectable proteins than in the other samples. Thus the comparison was performed on 15 control and 16 prostate cancer samples. The significantly differentially expressed proteins are summarized in Table 1, detailing the different annotations for the proteins. The vast majority of these proteins (221) were up-regulated in exosomes from prostate cancer, while a few (25) were down-regulated. 
     A ratio based on precursor ion intensity for top 3 total ion chromatograms (TOP3TIC) showing the enrichment of proteins in prostate cancer samples was calculated. The protein level of the samples was validated by pooling them into three sets of patient exosomes (PAT) and three sets of control exosomes (CTR) that were then subjected to LC/MS/MS with internal standard (iBAQ-intensity based absolute quantification) (Rosenberger G, Ludwig C, Rost H L, Aebersold R, &amp; Malmstrom L (2014) aLFQ: an R-package for estimating absolute protein quantities from label-free LC-MS/MS proteomics data.  Bioinformatics.,  30, 2511-2513). The ratios PAT versus CTR (iBAQ) are presented in Table 2 (bold: significant difference p&lt;0.05 in both analyses; normal font: significant only in discovery analysis). For the 21 proteins that were not detected in the validation study, the ratio obtained in the discovery analysis (TOP3TIC ratio) is shown in italics. The abundance of the proteins (ppm of total proteome) is also shown in Table 2, along with the number of patient or control samples in which the protein was confidently detected. Abundance values denote amounts found in the highest expression group (PAT for proteins overexpressed in prostate cancer, CTR for proteins underexpressed in prostate cancer). 
     A particularly good biomarker is characterized by having a high specificity and sensitivity for a specific condition. To identify the most promising biomarker candidates among the differentially expressed proteins, a tentative diagnostic call threshold was set for each individual protein to ensure 100% specificity (no erroneous positive call for CTR samples). The associated sensitivity levels were then calculated, and are detailed in Table 2 for each biomarker candidate. The proteins were analysed according to how many of the following criteria they met: (1) detected in validation study, (2) significantly altered in the validation study, (3) sensitivity above 40%, and (4) ratio PAT versus CTR above 1.75. The proteins found in Table 2 are first sorted by the number of criteria that they passed (more to less) and then by sensitivity (high to low). In relation to criteria (4), it is biomarkers whose level is increased in prostate cancer patients (samples) versus control that can have a PAT (patient) versus CTR (control) ratio of above 1.75. Analogously, for biomarkers whose level is decreased in prostate cancer patients (samples) versus control, an analogous criteria (4) may be applied, in which there is at least 1.75 times less expression of the biomarker in PAT versus CTR. 
     It was found that 58 proteins passed all 4 criteria. Data displaying the diagnostic call associated with each marker and patient sample for this focus list of promising biomarkers, based on the abovementioned specificity-driven diagnostic threshold, is presented in Table 3. Interestingly, 17 of the biomarkers displayed individual sensitivities above 60%, of which the highest sensitivity, at 94%, was observed for Sequence 1 (Uniprot entry name TM256_HUMAN—see Table 2). 
     Scatterplots, displaying the range of values observed in the PAT and CTR samples, are shown for illustrative purposes for the three biomarkers with the highest individual sensitivities at the chosen threshold ( FIG. 1 ). For each sample type, PAT and CTR, the intensity (TOP3TIC) was plotted displaying the distribution along the y-axis. The iBAQ ratio and the number of samples (PAT and CTR) in which the protein was detected are also shown. 
     The proteomic profile of exosomes from the prostate cancer cell line PC-3 has previously been described (Sandvig K &amp; Llorente A (2012) Proteomic analysis of microvesicles released by the human prostate cancer cell line PC-3.  Mol. Cell Proteomics.,  11, M111.012914). Only 29 proteins from Table 2 and 7 proteins from the more focused biomarker candidate list in Table 3 were common to the previously defined list of PC-3 exosomal proteins. These common proteins are summarised in Table 4. 
     All the biomarkers with individual diagnostic sensitivity above 60% (Sequences 1-17, see Table 2)), with the exception of Sequences 4, 14 and 16, could be combined with one other biomarker from Table 3 to provide full sensitivity and specificity for a two-marker test (assuming a positive call for at least one marker is sufficient for an overall positive diagnostic call). Sequences 20, 35 and 56, although of lower individual sensitivity, could also be combined with only one more marker (Sequence 1) to provide the same full diagnostic accuracy. The same could be achieved for Sequences 4, 14 and 16 in various three-marker combinations. Some examples of the 2- and 3-marker combinations providing full differentiation between prostate cancer and control samples are listed in Table 5. Other appropriate combinations are easily derived from the data in Table 3. 
     Other appropriate combinations of markers can be derived from Table 3 by combining two or more of the markers in Table 3 (e.g. 2, 3, 4, 5 or 6 markers, preferably 2, 3 or 4 markers) that results in one or more of the patients (P) (preferably the majority of the patients, e.g. 9, 10, 11, 12, 13, 14, 15 or 16 of the patients, more preferably all of the patients) being associated with a positive call (as indicated by a “1” in Table 3) from at least one marker in the combination. Put another way, other appropriate combinations of markers (sequences/polypeptides) can be derived from Table 3 by combining two or more of the sequence rows (e.g. 2, 3, 4, 5 or 6 sequence rows, preferably 2, 3 or 4 sequence rows) such that the combination of said two or more sequence rows has at least one positive call (as indicated by “1”) in one or more patient columns (P) (preferably the majority of the patient columns, e.g. 9, 10, 11, 12, 13, 14, 15 or 16 of the patient columns, more preferably all of the patient columns). By way of example, sequences (markers) 12, 13 and 14 would be an appropriate three-marker combination as when sequence rows 12, 13 and 14 are combined there is at least one positive call (“1”) in all of the patient columns (P). 
     Although as few as 1, 2 or 3 of the abovementioned biomarkers may be sufficient, more markers may be added to increase technical robustness. Furthermore, for some applications, the overall diagnostic call threshold for an expanded panel comprising the abovementioned markers may be set to require more than one positive call for the individual markers within the panel. This will reduce the rate of false positive diagnostic calls. A test requiring two independently positive markers for an overall positive diagnostic call, can still achieve full sensitivity with a combination of only four markers (an illustrative example, combining Sequences 1, 2, 3 and 9, is shown in Table 5). The diagnostic input from the individual markers in a panel may also be incorporated in an algorithm to provide a score, to be compared to a diagnostic threshold score. 
     The abovementioned analysis was based on setting a diagnostic threshold to provide 100% specificity (i.e. all control patient samples would be below the set threshold). One could also envision setting the appropriate diagnostic threshold to ensure maximum combined sensitivity and specificity. Table 6 shows the top ranking protein markers (those with a combined sensitivity and specificity of at least 160%) when performing such analysis. This alternative focus list of potential biomarkers displays some differences from the focus list of Table 3, which was developed based on specificity-driven diagnostic thresholds. Among 11 proteins in table 6 with a combined sensitivity and specificity above 170%, four were not included in table 3; Vesicle-associated membrane protein 2, Prenylcysteine oxidase 1, Sorcin and Grancalcin. 
     Scatterplots, displaying the range of values observed in the PAT and CTR samples for these proteins, are provided in  FIG. 2 . Particularly noteworthy among these is Prenylcysteine oxidase 1, which has an extreme outlier among the control samples, which dramatically reduces the sensitivity when setting a specificity-driven threshold. Discarding this outlier, which could very well be a non-diagnosed case with underlying disease, the protein is a very good discriminator between control samples and confirmed prostate cancer patients. Thus, Prenylcysteine oxidase 1 as well as other proteins in table 6 can be used as diagnostic biomarkers, and may be combined in a diagnostic test with any of the abovementioned proteins. 
     Experimental Procedures 
     Urine Samples 
     Urine samples were collected either from healthy control (15 samples) or from prostate cancer patients (17 samples) the day before prostatectomy. Samples were collected during the morning and were processed within 2 hours. The urine pH and the presence of leukocytes, nitrites, proteins, glucose, ketones and blood were analyzed with a Combur 7  strip-Test strip in an Urysis1100 urine analyzer (Roche Diagnostics). Creatinine was measured with a creatinine urinary detection kit (Arbor assays). The collection of urine samples was approved by the Norwegian Regional Committees for medical and health research ethics. 
     Exosome Isolation 
     Urinary exosomes were isolated by serial centrifugation. Briefly, urine was centrifuged at 2,000 g for 15 min, and then at 10,000 g for 30 min discarding the pellet at each step. The exosomes present in the supernatant were then pelleted at 100,000 g for 70 min and washed with PBS. Exosomes were then resuspended again in PBS, filtrated through a 200 nm pore filter and pelleted at 100,000 g for 70 min. The pellet was resuspended in 50-100 μ1 PBS and stored at −80 ° C. 
     Protein Measurements 
     The amount of protein in exosomes was determined using a BCA assay kit (Pierce, Thermo Scientific) according to the manufacturer&#39;s instructions. BSA was used as standard protein. 
     In-solution Digestion of Exosomes 
     Exosomes (2 μg) in one volume of PBS were mixed with four volumes of cold acetone (with 1M HCl) and methanol at −20 ° C. The samples were centrifuged at 15,000× g for 15 min and the pellets were dried in a Speed-Vac instrument. Then, the pellets were dissolved in 50 μl of a fresh solution of 100 mM ammonium bicarbonate with 6 M urea, and subsequently reduced with 10 mM dithiothreitol at 30° C. for 30 min. The samples were then incubated with 25 mM iodoacetamide to alkylate exposed side chains for 1 h at room temperature away from light. The enzymatic digestion was initiated by adding 1 μg Lys-C to the samples and incubating them at 37° C. for 2 hours. Finally, 240 μl 50 mM ammonium bicarbonate with 10 μg trypsin was added and the samples were first incubated for 1 h at 37 ° C., followed by 15 h at 30° C. Prior to LC-MS analysis, formic acid (5 μl ) was added to the digested exosomes. 
     Mass Spectrometry Analyses 
     For MS analyses, the samples (one quarter of the volume, 0.5 μg) were injected into an Ultimate 3000 nanoLC system (Dionex, Sunnyvale Calif., USA) connected to a linear quadrupole ion trap-orbitrap (LTQ-Orbitrap XL) mass spectrometer (ThermoScientific, Bremen, Germany) equipped with a nanoelectrospray ion source. An Acclaim PepMap 100 column (C18, 3 μm, 100 Å) (Dionex) with a capillary of 25 cm bed length was used for separation by liquid chromatography. A flow rate of 300 nl/min was employed with a solvent gradient of 4% B to 60% B in 230 min. Solvent A was 0.1% formic acid, whereas aqueous 90% acetonitrile in 0.1% formic acid was used as solvent B. The mass spectrometer was operated in the data-dependent mode to automatically switch between Orbitrap-MS and. LTQ-MS/MS acquisition. Survey full scan MS spectra (from m/z 300 to 2000) were acquired in the Orbitrap with resolution R=60,000 at m/z 400 (after accumulation to a target of 500,000 charges in the LTQ). The method used allowed sequential isolation of the most intense ions, up to six, depending on signal intensity, for fragmentation on the linear ion trap using collision induced dissociation at a target value of 10,000 charges. 
     To validate the quantitative analyses for the complete data set, the samples (aliquots of the digested exosomes that were used in the previous analysis) were pooled into three sets of patient exosomes and three sets of controls (aliquots of digested exosomes and subjected to LC/MS/MS with internal standard (iBAQ-intensity based absolute quantification (Rosenberger G, Ludwig C, Rost H L, Aebersold R, &amp; Malmstrom L (2014) aLFQ: an R-package for estimating absolute protein quantities from label-free LC-MS/MS proteomics data.  Bioinformatics.,  30, 2511-2513). The samples were separated on the Dionex U3000 capillary/nano-HPLC system (Dionex, Sunnyvale, Calif.), which was directly interfaced with a Thermo Fisher Q Exactive Orbitrap mass spectrometer. The mass spectrometer was operated in the data-dependent acquisition mode using the Xcalibur 2.2 software. Single MS full-scan in the Orbitrap (300-1750 m/z, 70,000 resolution at m/z 200, AGC target 1e6, maximum injection time 20 ms) was followed by 10 data-dependent MS/MS scans in the Orbitrap after accumulation of 1e6 ions in the C-trap or an injection time of 120 ms (fixed injection time method) at 35,000 resolution (isolation width 2.0 or 3.0 mlz, underfill ratio 0.1%, dynamic exclusion 20 or 45 s) at 25 or 30% normalized collision energy. Proteins that were present only in 1 of the 3 sets were considered invalid. 
     Data Processing 
     Tandem mass spectra were extracted, charge state deconvoluted and deisotoped by [Peptide Finder] version [1.8.1]. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.4.0). Mascot was set up to search the UniProt database (selected for Homo sapiens, ver 14.05.2014 version, 20279 entries) assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 10.0 ppm. Carbamidomethyl of cysteine was specified in Mascot as a fixed modification. Oxidation of methionine, acetylation of the N-terminus and phosphorylation of serine, threonine and tyrosine were specified in Mascot as variable modifications. Scaffold (version Scaffold 4.3.2, Proteome Software Inc., Portland, Oreg.) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Peptide Prophet algorithm (Keller A, Nesvizhskii A I, Kolker E, &amp; Aebersold R (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem., 74, 5383-5392) with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 1 identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii, Al et al Anal. Chem. 2003;75(17):4646-58). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. MS/MS spectra from protein hits identified with only 1 peptide were investigated manually. 
     Statistics 
     For comparing datasets Fisher&#39;s exact test (CI 95%) was used to determine significant changes between the subproteomes of exosomes from patients and healthy controls. The label-free quantitative measurement of individual samples used both PSM (peptide spectra match) and TOP3TIC (top 3 precursor intensities from total ion chromatogram) and only protein hits significantly altered (p&lt;0.05) for both were considered. The suitability as biomarker for the candidate proteins were addressed by determining an intensity threshold in every sample. The intensity threshold was optimized to give maximum specificity and sensitivity of the test, i.e. to maximize the difference between true positives (TP&gt;FN) and false negatives (TN&gt;FP). This enabled us to produce a table displaying most promising candidates within the cohort. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 List of proteins found to be differently expressed in urinary exosomes from prostate cancer patients  
               
               
                 compared to normal controls 
               
            
           
           
               
               
               
               
            
               
                   
                 Uniprot 
                 Uniprot 
                   
               
               
                 Sequence 
                 Entry Name 
                 Acc. No. 
                 Full Protein Name 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 TM256_HUMAN 
                 Q8N2U0 
                 Transmembrane protein 256 
               
               
                 2 
                 ADIRF_HUMAN 
                 Q15847 
                 Adipogenesis regulatory factor 
               
               
                 3 
                 LTOR1_HUMAN 
                 Q6IAA8 
                 Ragulator complex protein LAMTOR1 
               
               
                 4 
                 PLSL_HUMAN 
                 P13796 
                 Plastin-2 
               
               
                 5 
                 RAB2A_HUMAN 
                 P61019 
                 Ras-related protein Rab-2A 
               
               
                 6 
                 RAB3B_HUMAN 
                 P20337 
                 Ras-related protein Rab-3B 
               
               
                 7 
                 RAB3D_HUMAN 
                 O95716 
                 Ras-related protein Rab-3D 
               
               
                 8 
                 RAB7A_HUMAN 
                 P51149 
                 Ras-related protein Rab-7a 
               
               
                 9 
                 VATL_HUMAN 
                 P27449 
                 V-type proton ATPase 16 kDa proteolipid subunit 
               
               
                 10 
                 STEA4_HUMAN 
                 Q687X5 
                 Metalloreductase STEAP4 
               
               
                 11 
                 PARK7_HUMAN 
                 Q99497 
                 Protein DJ-1 
               
               
                 12 
                 S1OOP_HUMAN 
                 P25815 
                 Protein S100-P 
               
               
                 13 
                 SYTL4_HUMAN 
                 Q96C24 
                 Synaptotagmin-like protein 4 
               
               
                 14 
                 ARL8B_HUMAN 
                 Q9NVJ2 
                 ADP-ribosylation factor-like protein 8B 
               
               
                 15 
                 MYCT_HUMAN 
                 Q96QE2 
                 Proton myo-inositol cotransporter 
               
               
                 16 
                 RAB6A_HUMAN 
                 P20340 
                 Ras-related protein Rab-6A 
               
               
                 17 
                 TSN6_HUMAN 
                 O43657 
                 Tetraspanin-6 
               
               
                 18 
                 CLD10_HUMAN 
                 P78369 
                 Claudin-10 
               
               
                 19 
                 CLD2_HUMAN 
                 P57739 
                 Claudin-2 
               
               
                 20 
                 CLD3_HUMAN 
                 O15551 
                 Claudin-3 
               
               
                 21 
                 GMDS_HUMAN 
                 O60547 
                 GDP-mannose 4.6 dehydratase 
               
               
                 22 
                 GNPI1_HUMAN 
                 P46926 
                 Glucosamine-6-phosphate isomerase 1 
               
               
                 23 
                 SCRB2_HUMAN 
                 Q14108 
                 Lysosome membrane protein 2 
               
               
                 24 
                 MFS12_HUMAN 
                 Q6NUT3 
                 Major facilitator superfamily domain-containing protein 12 
               
               
                 25 
                 MELPH_HUMAN 
                 Q9BV36 
                 Melanophilin 
               
               
                 26 
                 SPRE_HUMAN 
                 P35270 
                 Sepiapterin reductase 
               
               
                 27 
                 TXD17_HUMAN 
                 Q9BRA2 
                 Thioredoxin domain-containing protein 17 
               
               
                 28 
                 BDH2_HUMAN 
                 Q9BUT1 
                 3-hydroxybutyrate dehydrogenase type 2 
               
               
                 29 
                 CALM_HUMAN 
                 P62158 
                 Calmodulin 
               
               
                 30 
                 CBPQ_HUMAN 
                 Q9Y646 
                 Carboxypeptidase Q 
               
               
                 31 
                 FLOT2_HUMAN 
                 Q14254 
                 Flotillin-2 
               
               
                 32 
                 LG3BP_HUMAN 
                 Q08380 
                 Galectin-3-binding protein 
               
               
                 33 
                 P2RX4_HUMAN 
                 Q99571 
                 P2X purinoceptor 4 
               
               
                 34 
                 DOP2_HUMAN 
                 Q9Y3R5 
                 Protein dopey-2 
               
               
                 35 
                 S10A6_HUMAN 
                 P06703 
                 Protein S100-A6 
               
               
                 36 
                 RAB35_HUMAN 
                 Q15286 
                 Ras-related protein Rab-35 
               
               
                 37 
                 PP2AA_HUMAN 
                 P67775 
                 Serine/threonine-protein phosphatase 2A catalytic subunit 
               
               
                   
                   
                   
                 alpha isoform 
               
               
                 38 
                 DNPH1_HUMAN 
                 Q43598 
                 2′-deoxynucleoside 5′-phosphate N-hydrolase 1 
               
               
                 39 
                 ASAH1_HUMAN 
                 Q13510 
                 Acid ceramidase 
               
               
                 40 
                 CALB1_HUMAN 
                 P05937 
                 Calbindin 
               
               
                 41 
                 CD59_HUMAN 
                 P13987 
                 CD59 glycoprotein 
               
               
                 42 
                 CD81_HUMAN 
                 P60033 
                 CD81 antigen 
               
               
                 43 
                 CY561_HUMAN 
                 P49447 
                 Cytochrome b561 
               
               
                 44 
                 ENOPH_HUMAN 
                 Q9UHY7 
                 Enolase-phosphatase E1 
               
               
                 45 
                 GOLP3_HUMAN 
                 Q9H4A6 
                 Golgi phosphoprotein 3 
               
               
                 46 
                 NICA_HUMAN 
                 Q92542 
                 Nicastrin 
               
               
                 47 
                 CPVL_HUMAN 
                 Q9H3G5 
                 Probable serine carboxypeptidase CPVL 
               
               
                 48 
                 LTOR5_HUMAN 
                 O43504 
                 Ragulator complex protein LAMTOR5 
               
               
                 49 
                 RB27B_HUMAN 
                 O00194 
                 Ras-related protein Rab-27B 
               
               
                 50 
                 SCAM2_HUMAN 
                 O15127 
                 Secretory carrier-associated membrane protein 2 
               
               
                 51 
                 SPSY_HUMAN 
                 P52788 
                 Spermine synthase 
               
               
                 52 
                 SKP1_HUMAN 
                 P63208 
                 S-phase kinase-associated protein 1 
               
               
                 53 
                 TM753_HUMAN 
                 Q9NS93 
                 Transmembrane 7 superfamily member 3 
               
               
                 54 
                 TPD52_HUMAN 
                 P55327 
                 Tumor protein D52 
               
               
                 55 
                 UB2V2_HUMAN 
                 Q15819 
                 Ubiquitin-conjugating enzyme E2 variant 2 
               
               
                 56 
                 UGDH_HUMAN 
                 O60701 
                 UDP-glucose 6-dehydrogenase 
               
               
                 57 
                 ZA2G_HUMAN 
                 P25311 
                 Zinc-alpha-2-glycoprotein 
               
               
                 58 
                 GDPD3_HUMAN 
                 Q7L5L3 
                 Glycerophosphodiester phosphodiesterase domain- 
               
               
                   
                   
                   
                 containing protein 3 
               
               
                 59 
                 EFHD2_HUMAN 
                 Q96C19 
                 EF-hand domain-containing protein D2 
               
               
                 60 
                 RAB14_HUMAN 
                 P61106 
                 Ras-related protein Rab-14 
               
               
                 61 
                 NIT2_HUMAN 
                 Q9NQR4 
                 Omega-amidase NIT2 
               
               
                 62 
                 ACTN1_HUMAN 
                 P12814 
                 Alpha-actinin-1 
               
               
                 63 
                 MOT5_HUMAN 
                 O15374 
                 Monocarboxylate transporter 5 
               
               
                 64 
                 RAB12_HUMAN 
                 Q6IQ22 
                 Ras-related protein Rab-12 
               
               
                 65 
                 RAB8A_HUMAN 
                 P61006 
                 Ras-related protein Rab-8A 
               
               
                 66 
                 TM63A_HUMAN 
                 O94886 
                 Transmembrane protein 63A 
               
               
                 67 
                 B2MG_HUMAN 
                 P61769 
                 Beta-2-microglobulin 
               
               
                 68 
                 VA0D1_HUMAN 
                 P61421 
                 V-type proton ATPase subunit d 1 
               
               
                 69 
                 LPP1_HUMAN 
                 O14494 
                 Lipid phosphate phosphohydrolase 1 
               
               
                 70 
                 GP155_HUMAN 
                 Q7Z3F1 
                 Integral membrane protein GPR155 
               
               
                 71 
                 1433S_HUMAN 
                 P31947 
                 14-3-3 protein sigma 
               
               
                 72 
                 LRSM1_HUMAN 
                 Q6UWE0 
                 E3 ubiquitin-protein ligase LRSAM1 
               
               
                 73 
                 DMA_HUMAN 
                 P28067 
                 HLA class 11 histocompatibility antigen. DM alpha chain 
               
               
                 74 
                 RAB9A_HUMAN 
                 P51151 
                 Ras-related protein Rab-9A 
               
               
                 75 
                 SRC8_HUMAN 
                 Q14247 
                 Src substrate cortactin 
               
               
                 76 
                 AQP7_HUMAN 
                 O14520 
                 Aquaporin-7 
               
               
                 77 
                 SYUG_HUMAN 
                 O76070 
                 Gamma-synuclein 
               
               
                 78 
                 1433T_HUMAN 
                 P27348 
                 14-3-3 protein theta 
               
               
                 79 
                 AATC_HUMAN 
                 P17174 
                 Aspartate aminotransferase. cytoplasmic 
               
               
                 80 
                 CLIC3_HUMAN 
                 O95833 
                 Chloride intracellular channel protein 3 
               
               
                 81 
                 DEST_HUMAN 
                 P60981 
                 Destrin 
               
               
                 82 
                 RASH_HUMAN 
                 P01112 
                 GTPase HRas 
               
               
                 83 
                 PTGR2_HUMAN 
                 Q8N8N7 
                 Prostaglandin reductase 2 
               
               
                 84 
                 TCPE_HUMAN 
                 P48643 
                 T-complex protein 1 subunit epsilon 
               
               
                 85 
                 ITIH4_HUMAN 
                 Q14624 
                 Inter-alpha-trypsin inhibitor heavy chain H4 
               
               
                 86 
                 AL1A3_HUMAN 
                 P47895 
                 Aldehyde dehydrogenase family 1 member A3 
               
               
                 87 
                 ANXA3_HUMAN 
                 P12429 
                 Annexin A3 
               
               
                 88 
                 CLN3_HUMAN 
                 Q13286 
                 Battenin 
               
               
                 89 
                 CATD_HUMAN 
                 P07339 
                 Cathepsin D 
               
               
                 90 
                 DDAH1_HUMAN 
                 O94760 
                 N(G).N(G)-dimethylarginine dimethylaminohydrolase 1 
               
               
                 91 
                 NPDC1_HUMAN 
                 Q9NQX5 
                 Neural proliferation differentiation and control protein 1 
               
               
                 92 
                 SAP_HUMAN 
                 P07602 
                 Proactivator polypeptide 
               
               
                 93 
                 KLK3_HUMAN 
                 P07288 
                 Prostate-specific antigen 
               
               
                 94 
                 LFG3_HUMAN 
                 Q969X1 
                 Protein lifeguard 3 
               
               
                 95 
                 NIBAN_HUMAN 
                 Q9BZQ8 
                 Protein Niban 
               
               
                 96 
                 SPNS1_HUMAN 
                 Q9H2V7 
                 Protein spinster homolog 1 
               
               
                 97 
                 LTOR2_HUMAN 
                 Q9Y2Q5 
                 Ragulator complex protein LAMTOR2 
               
               
                 98 
                 LTOR3_HUMAN 
                 Q9UHA4 
                 Ragulator complex protein LAMTOR3 
               
               
                 99 
                 SYT7_HUMAN 
                 O43581 
                 Synaptotagmin-7 
               
               
                 100 
                 T106B_HUMAN 
                 Q9NUM4 
                 Transmembrane protein 106B 
               
               
                 101 
                 MYO5C_HUMAN 
                 Q9NQX4 
                 Unconventional myosin-Vc 
               
               
                 102 
                 VAMP2_HUMAN 
                 P63027 
                 Vesicle-associated membrane protein 2 
               
               
                 103 
                 VATF_HUMAN 
                 Q16864 
                 V-type proton ATPase subunit F 
               
               
                 104 
                 AKA7A_HUMAN 
                 O43687 
                 A-kinase anchor protein 7 isoforms alpha and beta 
               
               
                 105 
                 ARSF_HUMAN 
                 P54793 
                 Arylsulfatase F 
               
               
                 106 
                 JIP4_HUMAN 
                 O60271 
                 C-Jun-amino-terminal kinase-interacting protein 4 
               
               
                 107 
                 DMBT1_HUMAN 
                 Q9UGM3 
                 Deleted in malignant brain tumors 1 protein 
               
               
                 108 
                 GMFB_HUMAN 
                 P60983 
                 Glia maturation factor beta 
               
               
                 109 
                 FOLH1_HUMAN 
                 Q04609 
                 Glutamate carboxypeptidase 2 
               
               
                 110 
                 GSHB_HUMAN 
                 P48637 
                 Glutathione synthetase 
               
               
                 111 
                 HPCL1_HUMAN 
                 P37235 
                 Hippocalcin-like protein 1 
               
               
                 112 
                 MARCS_HUMAN 
                 P29966 
                 Myristoylated alanine-rich C-kinase substrate 
               
               
                 113 
                 NNRE_HUMAN 
                 Q8NCW5 
                 NAD(P)H-hydrate epimerase 
               
               
                 114 
                 NAPSA_HUMAN 
                 O96009 
                 Napsin-A 
               
               
                 115 
                 AGM1_HUMAN 
                 O95394 
                 Phosphoacetylglucosamine mutase 
               
               
                 116 
                 AT8A1_HUMAN 
                 Q9Y2Q0 
                 Probable phospholipid-transporting ATPase IA 
               
               
                 117 
                 QOR_HUMAN 
                 Q08257 
                 Quinone oxidoreductase 
               
               
                 118 
                 RET5_HUMAN 
                 P82980 
                 Retinol-binding protein 5 
               
               
                 119 
                 SERC2_HUMAN 
                 Q96SA4 
                 Serine incorporator 2 
               
               
                 120 
                 S15A1_HUMAN 
                 P46059 
                 Solute carrier family 15 member 1 
               
               
                 121 
                 SORCN_HUMAN 
                 P30626 
                 Sorcin 
               
               
                 122 
                 SUIS_HUMAN 
                 P14410 
                 Sucrase-isomaltase. intestinal 
               
               
                 123 
                 ARPC4_HUMAN 
                 P59998 
                 Actin-related protein 2/3 complex subunit 4 
               
               
                 124 
                 ARPC5_HUMAN 
                 O15511 
                 Actin-related protein 2/3 complex subunit 5 
               
               
                 125 
                 APT_HUMAN 
                 P07741 
                 Adenine phosphoribosyltransferase 
               
               
                 126 
                 ABRAL_HUMAN 
                 Q9P1F3 
                 Costars family protein ABRACL 
               
               
                 127 
                 IGHA1_HUMAN 
                 P01876 
                 Ig alpha-1 chain C region 
               
               
                 128 
                 IFM3_HUMAN 
                 Q01628 
                 Interferon-induced transmembrane protein 3 
               
               
                 129 
                 TRFL_HUMAN 
                 P02788 
                 Lactotransferrin 
               
               
                 130 
                 PNPH_HUMAN 
                 P00491 
                 Purine nucleoside phosphorylase 
               
               
                 131 
                 STX134_HUMAN 
                 Q6ZWJ1 
                 Syntaxin-binding protein 4 
               
               
                 132 
                 TSN8_HUMAN 
                 P19075 
                 Tetraspanin-8 
               
               
                 133 
                 VPS25_HUMAN 
                 Q9BRG1 
                 Vacuolar protein-sorting-associated protein 25 
               
               
                 134 
                 VDAC1_HUMAN 
                 P21796 
                 Voltage-dependent anion-selective channel protein 1 
               
               
                 135 
                 CO6A1_HUMAN 
                 P12109 
                 Collagen alpha-1(VI) chain 
               
               
                 136 
                 STAM1_HUMAN 
                 Q92783 
                 Signal transducing adapter molecule 1 
               
               
                 137 
                 CD9_HUMAN 
                 P21926 
                 CD9 antigen 
               
               
                 138 
                 FLOT1_HUMAN 
                 O75955 
                 Flotillin-1 
               
               
                 139 
                 GRAN_HUMAN 
                 P28676 
                 Grancalcin 
               
               
                 140 
                 GMPPB_HUMAN 
                 Q9Y5P6 
                 Mannose-1-phosphate guanyltransferase beta 
               
               
                 141 
                 PSA7_HUMAN 
                 O14818 
                 Proteasome subunit alpha type-7 
               
               
                 142 
                 RAB18_HUMAN 
                 Q9NP72 
                 Ras-related protein Rab-18 
               
               
                 143 
                 VP37C_HUMAN 
                 A5D8V6 
                 Vacuolar protein sorting-associated protein 37C 
               
               
                 144 
                 STAM2_HUMAN 
                 O75886 
                 Signal transducing adapter molecule 2 
               
               
                 145 
                 GP2_HUMAN 
                 P55259 
                 Pancreatic secretory granule membrane major glycoprotein 
               
               
                   
                   
                   
                 GP2 
               
               
                 146 
                 PLCG2_HUMAN 
                 P16885 
                 1-phosphatidylinositol 4.5-bisphosphate phosphodiesterase 
               
               
                   
                   
                   
                 gamma-2 
               
               
                 147 
                 LAMP2_HUMAN 
                 P13473 
                 Lysosome-associated membrane glycoprotein 2 
               
               
                 148 
                 CRCT1_HUMAN 
                 Q9UGL9 
                 Cysteine-rich C-terminal protein 1 
               
               
                 149 
                 IPSP_HUMAN 
                 P05154 
                 Plasma serine protease inhibitor 
               
               
                 150 
                 LCK_HUMAN 
                 P06239 
                 Tyrosine-protein kinase Lck 
               
               
                 151 
                 LTOR4 
                 Q0VGL1 
                 Ragulator complex protein LAMTOR4 
               
               
                 152 
                 SCAM1_HUMAN 
                 O15126 
                 Secretory carrier-associated membrane protein 1 
               
               
                 153 
                 FKB1A_HUMAN 
                 P62942 
                 Peptidyl-prolyl cis-trans isomerase FKBP1A 
               
               
                 154 
                 T176A_HUMAN 
                 Q96HP8 
                 Transmembrane protein 176A 
               
               
                 155 
                 TYB4_HUMAN 
                 P62328 
                 Thymosin beta-4 
               
               
                 156 
                 HDHD2_HUMAN 
                 Q9H0R4 
                 Haloacid dehalogenase-like hydrolase domain-containing 
               
               
                   
                   
                   
                 protein 2 
               
               
                 157 
                 CDC42_HUMAN 
                 P60953 
                 Cell division control protein 42 homolog 
               
               
                 158 
                 RAB17_HUMAN 
                 Q9H0T7 
                 Ras-related protein Rab-17 
               
               
                 159 
                 CLIC6_HUMAN 
                 Q96NY7 
                 Chloride intracellular channel protein 6 
               
               
                 160 
                 CTL4_HUMAN 
                 Q53GD3 
                 Choline transporter-like protein 4 
               
               
                 161 
                 BLVRB_HUMAN 
                 P30043 
                 Flavin reductase (NADPH) 
               
               
                 162 
                 RAB10_HUMAN 
                 P61026 
                 Ras-related protein Rab-10 
               
               
                 163 
                 HEBP2_HUMAN 
                 Q9Y5Z4 
                 Heme-binding protein 2 
               
               
                 164 
                 FABP5_HUMAN 
                 Q01469 
                 Fatty acid-binding protein. epidermal 
               
               
                 165 
                 SMIM5_HUMAN 
                 Q71RC9 
                 Small integral membrane protein 5 
               
               
                 166 
                 LAP4A_HUMAN 
                 Q15012 
                 Lysosomal-associated transmembrane protein 4A 
               
               
                 167 
                 PHYD1_HUMAN 
                 Q5SRE7 
                 Phytanoyl-CoA dioxygenase domain-containing protein 1 
               
               
                 168 
                 PSA5_HUMAN 
                 P28066 
                 Proteasome subunit alpha type-5 
               
               
                 169 
                 CALL3_HUMAN 
                 P27482 
                 Calmodulin-like protein 3 
               
               
                 170 
                 PSN1_HUMAN 
                 P49768 
                 Presenilin-1 
               
               
                 171 
                 NQ02_HUMAN 
                 P16083 
                 Ribosyldihydronicotinamide dehydrogenase [quinone] 
               
               
                 172 
                 TCTP_HUMAN 
                 P13693 
                 Translationally-control led tumor protein 
               
               
                 173 
                 LAMP1_HUMAN 
                 P11279 
                 Lysosome-associated membrane glycoprotein 1 
               
               
                 174 
                 CD38_HUMAN 
                 P28907 
                 ADP-ribosyl cyclase 1 
               
               
                 175 
                 MTPN_HUMAN 
                 P58546 
                 Myotrophin 
               
               
                 176 
                 DYL2_HUMAN 
                 Q96FJ2 
                 Dynein light chain 2. cytoplasmic 
               
               
                 177 
                 DHPR_HUMAN 
                 P09417 
                 Dihydropteridine reductase 
               
               
                 178 
                 NADC_HUMAN 
                 Q15274 
                 Nicotinate-nucleotide pyrophosphorylase [carboxylating] 
               
               
                 179 
                 CC50A_HUMAN 
                 Q9NV96 
                 Cell cycle control protein 50A 
               
               
                 180 
                 PMVK_HUMAN 
                 Q15126 
                 Phosphomevalonate kinase 
               
               
                 181 
                 IF4H_HUMAN 
                 Q15056 
                 Eukaryotic translation initiation factor 4H 
               
               
                 182 
                 TTYH3_HUMAN 
                 Q9C0H2 
                 Protein tweety homolog 3 
               
               
                 183 
                 NPT2B_HUMAN 
                 O95436 
                 Sodium-dependent phosphate transport protein 2B 
               
               
                 184 
                 LAC2_HUMAN (+1)  
                 P0CG05 
                 Ig lambda-2 chain C regions 
               
               
                 185 
                 RABP2_HUMAN 
                 P29373 
                 Cellular retinoic acid-binding protein 2 
               
               
                 186 
                 CUTA_HUMAN 
                 O60888 
                 Protein CutA 
               
               
                 187 
                 PSA4_HUMAN 
                 P25789 
                 Proteasome subunit alpha type-4 
               
               
                 188 
                 S35F6_HUMAN 
                 Q8N357 
                 Solute carrier family 35 member F6 
               
               
                 189 
                 HEM2_HUMAN 
                 P13716 
                 Delta-aminolevulinic acid dehydratase 
               
               
                 190 
                 DCXR_HUMAN 
                 Q7Z4W1 
                 L-xylulose reductase 
               
               
                 191 
                 UPK1A_HUMAN 
                 O00322 
                 Uroplakin-1a 
               
               
                 192 
                 SPR1A_HUMAN 
                 P35321 
                 Cornifin-A 
               
               
                 193 
                 ZN185_HUMAN 
                 O15231 
                 Zinc finger protein 185 
               
               
                 194 
                 TMM8A_HUMAN 
                 Q9HCN3 
                 Transmembrane protein 8A 
               
               
                 195 
                 PCYOX_HUMAN 
                 Q9UHG3 
                 Prenylcysteine oxidase 1 
               
               
                 196 
                 LYSC_HUMAN 
                 P61626 
                 Lysozyme C 
               
               
                 197 
                 PALM_HUMAN 
                 O75781 
                 Paralemmin-1 
               
               
                 198 
                 CEAM6_HUMAN 
                 P40199 
                 Carcinoembryonic antigen-related cell adhesion molecule 6 
               
               
                 199 
                 SC5A1_HUMAN 
                 P13866 
                 Sodium/glucose cotransporter 1 
               
               
                 200 
                 PTGR1_HUMAN 
                 Q14914 
                 Prostaglandin reductase 1 
               
               
                 201 
                 S10A9_HUMAN 
                 P06702 
                 Protein S100-A9 
               
               
                 202 
                 MILK1_HUMAN 
                 Q8N3F8 
                 MICAL-like protein 1 
               
               
                 203 
                 ABHEB_HUMAN 
                 Q96IU4 
                 Alpha/beta hydrolase domain-containing protein 14B 
               
               
                 204 
                 AQP2_HUMAN 
                 P41181 
                 Aquaporin-2 
               
               
                 205 
                 GSTP1_HUMAN 
                 P09211 
                 Glutathione 5-transferase P 
               
               
                 206 
                 ZDHC1_HUMAN 
                 Q8WTX9 
                 Probable palmitoyltransferase ZDHHC1 
               
               
                 207 
                 RAB8B_HUMAN 
                 Q92930 
                 Ras-related protein Rab-8B 
               
               
                 208 
                 TMPS2_HUMAN 
                 O15393 
                 Transmembrane protease serine 2 
               
               
                 209 
                 RAB1B_HUMAN 
                 Q9H0U4 
                 Ras-related protein Rab-1B 
               
               
                 210 
                 RAB1A_HUMAN 
                 P62820 
                 Ras-related protein Rab-1A 
               
               
                 211 
                 RAB43_HUMAN 
                 Q86YS6 
                 Ras-related protein Rab-43 
               
               
                 212 
                 SNG2_HUMAN 
                 O43760 
                 Synaptogyrin-2 
               
               
                 213 
                 2B1F_HUMAN 
                 P01911 
                 HLA class II histocompatibility antigen. DRB1-15 beta  
               
               
                   
                   
                   
                 chain 
               
               
                 214 
                 NCUG1_HUMAN 
                 Q8WWB7 
                 Lysosomal protein NCU-G1 
               
               
                 215 
                 CAB39_HUMAN 
                 Q9Y376 
                 Calcium-binding protein 39 
               
               
                 216 
                 DYN2_HUMAN 
                 P50570 
                 Dynamin-2 
               
               
                 217 
                 C42S2_HUMAN 
                 Q9NRR3 
                 CDC42 small effector protein 2 
               
               
                 218 
                 FRIH_HUMAN 
                 P02794 
                 Ferritin heavy chain 
               
               
                 219 
                 S35F2_HUMAN 
                 Q8IXU6 
                 Solute carrier family 35 member F2 
               
               
                 220 
                 PNKD_HUMAN 
                 Q8N490 
                 Probable hydrolase PNKD 
               
               
                 221 
                 CATZ_HUMAN 
                 Q9UBR2 
                 Cathepsin Z 
               
               
                 222 
                 TBB2B_HUMAN 
                 Q9BVA1 
                 Tubulin beta-2B chain 
               
               
                 223 
                 TSTD1_HUMAN 
                 Q8NFU3 
                 Thiosulfate sulfurtransferase/rhodanese-like domain- 
               
               
                   
                   
                   
                 containing protein 1 
               
               
                 224 
                 MK01_HUMAN 
                 P28482 
                 Mitogen-activated protein kinase 1 
               
               
                 225 
                 ADHX_HUMAN 
                 P11766 
                 Alcohol dehydrogenase class-3 
               
               
                 226 
                 PPAC_HUMAN 
                 P24666 
                 Low molecular weight phosphotyrosine protein phosphatase 
               
               
                 227 
                 ANXA4_HUMAN 
                 P09525 
                 Annexin A4 
               
               
                 228 
                 SEPT2_HUMAN 
                 Q15019 
                 Septin-2 
               
               
                 229 
                 GSTM3_HUMAN 
                 P21266 
                 Glutathione 5-transferase Mu 3 
               
               
                 230 
                 BRK1_HUMAN 
                 Q8WUW1 
                 Protein BRICK1 
               
               
                 231 
                 PSB2_HUMAN 
                 P49721 
                 Proteasome subunit beta type-2 
               
               
                 232 
                 UBE2K_HUMAN 
                 P61086 
                 Ubiquitin-conjugating enzyme E2 K 
               
               
                 233 
                 S10A1_HUMAN 
                 P23297 
                 Protein S100-A1 
               
               
                 234 
                 MAP1A_HUMAN 
                 P78559 
                 Microtubule-associated protein 1A 
               
               
                 235 
                 GSTM1_HUMAN 
                 P09488 
                 Glutathione S-transferase Mu 1 
               
               
                 236 
                 MMP24_HUMAN 
                 Q9Y5R2 
                 Matrix metalloproteinase-24 
               
               
                 237 
                 SIM22_HUMAN 
                 K7EJ46 
                 Small integral membrane protein 22 
               
               
                 238 
                 HGNAT_HUMAN 
                 Q68CP4 
                 Heparan-alpha-glucosaminide N-acetyltransferase 
               
               
                 239 
                 SARG_HUMAN 
                 Q9BW04 
                 Specifically androgen-regulated gene protein 
               
               
                 240 
                 ABI1_HUMAN 
                 Q8IZP0 
                 Abl interactor 1 
               
               
                 241 
                 CF132_HUMAN 
                 Q5T0Z8 
                 Uncharacterized protein C6orf132 
               
               
                 242 
                 ARF5_HUMAN 
                 P84085 
                 ADP-ribosylation factor 5 
               
               
                 243 
                 IDHC_HUMAN 
                 O75874 
                 Isocitrate dehydrogenase [NADP] cytoplasmic 
               
               
                 244 
                 GLTP_HUMAN 
                 Q9NZD2 
                 Glycolipid transfer protein 
               
               
                 245 
                 TPM4_HUMAN 
                 P67936 
                 Tropomyosin alpha-4 chain 
               
               
                 246 
                 NRAM2_HUMAN 
                 P49281 
                 Natural resistance-associated macrophage protein 2 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Diagnostic potential of differentially expressed proteins. 
               
            
           
           
               
               
               
               
            
               
                   
                 Data from 
                 Data from 
                   
               
               
                   
                 discovery analysis 
                 validation analysis 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 #Present 
                 #Present 
                 Validated 
                 Abundance 
                   
               
               
                   
                 Uniprot 
                 Uniprot 
                   
                 in CTR 
                 in PAT 
                 iBAQ ratio 
                 (ppm of total 
                 #Passed 
               
               
                 Sequence 
                 Entry Name 
                 Acc. No. 
                 Sensitivity 
                 (of 15) 
                 (of 16) 
                 PAT:CTR 
                 proteome) 
                 criteria 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 TM256_HUMAN 
                 Q8N2U0 
                 94% 
                 5 
                 16 
                 
                   140.39 
                 
                 4324 
                 
                   4 
                 
               
               
                 2 
                 ADIRF_HUMAN 
                 Q15847 
                 81% 
                 4 
                 15 
                 
                   18.99 
                 
                 369 
                 
                   4 
                 
               
               
                 3 
                 LTOR1_HUMAN 
                 Q6IAA8 
                 81% 
                 4 
                 16 
                 
                   22.98 
                 
                 201 
                 
                   4 
                 
               
               
                 4 
                 PLSL_HUMAN 
                 P13796 
                 75% 
                 14 
                 16 
                 
                   3.15 
                 
                 256 
                 
                   4 
                 
               
               
                 5 
                 RAB2A_HUMAN 
                 P61019 
                 75% 
                 14 
                 16 
                 
                   3.55 
                 
                 1083 
                 
                   4 
                 
               
               
                 6 
                 RAB3B_HUMAN 
                 P20337 
                 75% 
                 15 
                 16 
                 
                   2.69 
                 
                 1138 
                 
                   4 
                 
               
               
                 7 
                 RAB3D_HUMAN 
                 O95716 
                 75% 
                 15 
                 16 
                 
                   2.24 
                 
                 2340 
                 
                   4 
                 
               
               
                 8 
                 RAB7A_HUMAN 
                 P51149 
                 75% 
                 15 
                 16 
                 
                   3.26 
                 
                 2317 
                 
                   4 
                 
               
               
                 9 
                 VATL_HUMAN 
                 P27449 
                 75% 
                 0 
                 12 
                 
                   3.55 
                 
                 861 
                 
                   4 
                 
               
               
                 10 
                 STEA4_HUMAN 
                 Q687X5 
                 69% 
                 14 
                 16 
                 
                   2.97 
                 
                 953 
                 
                   4 
                 
               
               
                 11 
                 PARK7_HUMAN 
                 Q99497 
                 69% 
                 15 
                 16 
                 
                   1.92 
                 
                 957 
                 
                   4 
                 
               
               
                 12 
                 S100P_HUMAN 
                 P25815 
                 69% 
                 14 
                 15 
                 
                   1.84 
                 
                 1351 
                 
                   4 
                 
               
               
                 13 
                 SYTL4_HUMAN 
                 Q96C24 
                 69% 
                 5 
                 12 
                 
                   3.08 
                 
                 91 
                 
                   4 
                 
               
               
                 14 
                 ARL8B_HUMAN 
                 Q9NVJ2 
                 63% 
                 13 
                 15 
                 
                   2.79 
                 
                 49 
                 
                   4 
                 
               
               
                 15 
                 MYCT_HUMAN 
                 Q96QE2 
                 63% 
                 2 
                 11 
                 
                   2.66 
                 
                 100 
                 
                   4 
                 
               
               
                 16 
                 RAB6A_HUMAN 
                 P20340 
                 63% 
                 10 
                 16 
                 
                   3.36 
                 
                 240 
                 
                   4 
                 
               
               
                 17 
                 TSN6_HUMAN 
                 O43657 
                 63% 
                 9 
                 16 
                 
                   4.03 
                 
                 3067 
                 
                   4 
                 
               
               
                 18 
                 CLD10_HUMAN 
                 P78369 
                 56% 
                 7 
                 14 
                 
                   2.14 
                 
                 26 
                 
                   4 
                 
               
               
                 19 
                 CLD2_HUMAN 
                 P57739 
                 56% 
                 2 
                 12 
                 
                   3.00 
                 
                 69 
                 
                   4 
                 
               
               
                 20 
                 CLD3_HUMAN 
                 O15551 
                 56% 
                 1 
                 10 
                 
                   1.75 
                 
                 170 
                 
                   4 
                 
               
               
                 21 
                 GMDS_HUMAN 
                 O60547 
                 56% 
                 2 
                 12 
                 
                   2.45 
                 
                 16 
                 
                   4 
                 
               
               
                 22 
                 GNPI1_HUMAN 
                 P46926 
                 56% 
                 4 
                 13 
                 
                   15.51 
                 
                 44 
                 
                   4 
                 
               
               
                 23 
                 SCRB2_HUMAN 
                 Q14108 
                 56% 
                 15 
                 16 
                 
                   3.94 
                 
                 824 
                 
                   4 
                 
               
               
                 24 
                 MFS12_HUMAN 
                 Q6NUT3 
                 56% 
                 5 
                 15 
                 
                   8.07 
                 
                 65 
                 
                   4 
                 
               
               
                 25 
                 MELPH_HUMAN 
                 Q9BV36 
                 56% 
                 12 
                 16 
                 
                   2.26 
                 
                 151 
                 
                   4 
                 
               
               
                 26 
                 SPRE_HUMAN 
                 P35270 
                 56% 
                 10 
                 14 
                 
                   2.16 
                 
                 114 
                 
                   4 
                 
               
               
                 27 
                 TXD17_HUMAN 
                 Q9BRA2 
                 56% 
                 15 
                 16 
                 
                   2.35 
                 
                 288 
                 
                   4 
                 
               
               
                 28 
                 BDH2_HUMAN 
                 Q9BUT1 
                 50% 
                 14 
                 16 
                 
                   2.26 
                 
                 389 
                 
                   4 
                 
               
               
                 29 
                 CALM_HUMAN 
                 P62158 
                 50% 
                 15 
                 16 
                 
                   6.30 
                 
                 4764 
                 
                   4 
                 
               
               
                 30 
                 CBPQ_HUMAN 
                 Q9Y646 
                 50% 
                 1 
                 8 
                 
                   5.80 
                 
                 51 
                 
                   4 
                 
               
               
                 31 
                 FLOT2_HUMAN 
                 Q14254 
                 50% 
                 14 
                 16 
                 
                   2.89 
                 
                 541 
                 
                   4 
                 
               
               
                 32 
                 LG3BP_HUMAN 
                 Q08380 
                 50% 
                 15 
                 16 
                 
                   1.99 
                 
                 678 
                 
                   4 
                 
               
               
                 33 
                 P2RX4_HUMAN 
                 Q99571 
                 50% 
                 6 
                 13 
                 
                   2.36 
                 
                 76 
                 
                   4 
                 
               
               
                 34 
                 DOP2_HUMAN 
                 Q9Y3R5 
                 50% 
                 12 
                 16 
                 
                   2.99 
                 
                 218 
                 
                   4 
                 
               
               
                 35 
                 S10A6_HUMAN 
                 P06703 
                 50% 
                 15 
                 16 
                 
                   0.48 
                 
                 1853 
                 
                   4 
                 
               
               
                 36 
                 RAB35_HUMAN 
                 Q15286 
                 50% 
                 15 
                 16 
                 
                   0.57 
                 
                 245 
                 
                   4 
                 
               
               
                 37 
                 PP2AA_HUMAN 
                 P67775 
                 50% 
                 11 
                 14 
                 
                   16.91 
                 
                 41 
                 
                   4 
                 
               
               
                 38 
                 DNPH1_HUMAN 
                 O43598 
                 44% 
                 7 
                 14 
                 
                   2.26 
                 
                 117 
                 
                   4 
                 
               
               
                 39 
                 ASAH1_HUMAN 
                 Q13510 
                 44% 
                 15 
                 16 
                 
                   3.73 
                 
                 967 
                 
                   4 
                 
               
               
                 40 
                 CALB1_HUMAN 
                 P05937 
                 44% 
                 15 
                 16 
                 
                   2.02 
                 
                 1907 
                 
                   4 
                 
               
               
                 41 
                 CD59_HUMAN 
                 P13987 
                 44% 
                 15 
                 16 
                 
                   0.00 
                 
                 2 
                 
                   4 
                 
               
               
                 42 
                 CD81_HUMAN 
                 P60033 
                 44% 
                 13 
                 15 
                 
                   2.61 
                 
                 918 
                 
                   4 
                 
               
               
                 43 
                 CY561_HUMAN 
                 P49447 
                 44% 
                 1 
                 11 
                 
                   24.50 
                 
                 86 
                 
                   4 
                 
               
               
                 44 
                 ENOPH_HUMAN 
                 Q9UHY7 
                 44% 
                 1 
                 7 
                 
                   2.66 
                 
                 55 
                 
                   4 
                 
               
               
                 45 
                 GOLP3_HUMAN 
                 Q9H4A6 
                 44% 
                 1 
                 15 
                 
                   1.86 
                 
                 9 
                 
                   4 
                 
               
               
                 46 
                 NICA_HUMAN 
                 Q92542 
                 44% 
                 10 
                 16 
                 
                   2.37 
                 
                 170 
                 
                   4 
                 
               
               
                 47 
                 CPVL_HUMAN 
                 Q9H3G5 
                 44% 
                 1 
                 7 
                 
                   6.25 
                 
                 75 
                 
                   4 
                 
               
               
                 48 
                 LTOR5_HUMAN 
                 O43504 
                 44% 
                 0 
                 7 
                 
                   2.40 
                 
                 192 
                 
                   4 
                 
               
               
                 49 
                 RB27B_HUMAN 
                 O00194 
                 44% 
                 15 
                 16 
                 
                   2.24 
                 
                 1850 
                 
                   4 
                 
               
               
                 50 
                 SCAM2_HUMAN 
                 O15127 
                 44% 
                 0 
                 7 
                 
                   3.51 
                 
                 230 
                 
                   4 
                 
               
               
                 51 
                 SPSY_HUMAN 
                 P52788 
                 44% 
                 8 
                 14 
                 
                   2.46 
                 
                 143 
                 
                   4 
                 
               
               
                 52 
                 SKP1_HUMAN 
                 P63208 
                 44% 
                 10 
                 16 
                 
                   7.68 
                 
                 12 
                 
                   4 
                 
               
               
                 53 
                 TM7S3_HUMAN 
                 Q9NS93 
                 44% 
                 15 
                 15 
                 
                   4.15 
                 
                 198 
                 
                   4 
                 
               
               
                 54 
                 TPD52_HUMAN 
                 P55327 
                 44% 
                 0 
                 7 
                 
                   13.69 
                 
                 34 
                 
                   4 
                 
               
               
                 55 
                 UB2V2_HUMAN 
                 Q15819 
                 44% 
                 11 
                 15 
                 
                   2.10 
                 
                 289 
                 
                   4 
                 
               
               
                 56 
                 UGDH_HUMAN 
                 O60701 
                 44% 
                 13 
                 16 
                 
                   4.11 
                 
                 249 
                 
                   4 
                 
               
               
                 57 
                 ZA2G_HUMAN 
                 P25311 
                 44% 
                 8 
                 14 
                 
                   2.49 
                 
                 942 
                 
                   4 
                 
               
               
                 58 
                 GDPD3_HUMAN 
                 Q7L5L3 
                 41% 
                 2 
                 16 
                 
                   6.31 
                 
                 40 
                 
                   4 
                 
               
               
                 59 
                 EFHD2_HUMAN 
                 Q96C19 
                 63% 
                 13 
                 15 
                 1.75 
                 71 
                 
                   3 
                 
               
               
                 60 
                 RAB14_HUMAN 
                 P61106 
                 63% 
                 15 
                 16 
                 2.34 
                 975 
                 
                   3 
                 
               
               
                 61 
                 NIT2_HUMAN 
                 Q9NQR4 
                 63% 
                 13 
                 16 
                 
                   1.58 
                 
                 393 
                 
                   3 
                 
               
               
                 62 
                 ACTN1_HUMAN 
                 P12814 
                 56% 
                 8 
                 12 
                 
                   1.33 
                 
                 33652 
                 
                   3 
                 
               
               
                 63 
                 MOT5_HUMAN 
                 O15374 
                 56% 
                 0 
                 9 
                 
                   1.33 
                 
                 383 
                 
                   3 
                 
               
               
                 64 
                 RAB12_HUMAN 
                 Q6IQ22 
                 56% 
                 14 
                 15 
                 
                   1.57 
                 
                 22 
                 
                   3 
                 
               
               
                 65 
                 RAB8A_HUMAN 
                 P61006 
                 56% 
                 15 
                 16 
                 
                   1.24 
                 
                 323 
                 
                   3 
                 
               
               
                 66 
                 TM63A_HUMAN 
                 O94886 
                 56% 
                 13 
                 16 
                 
                   1.71 
                 
                 69 
                 
                   3 
                 
               
               
                 67 
                 B2MG_HUMAN 
                 P61769 
                 50% 
                 4 
                 9 
                 0.20 
                 13 
                 
                   3 
                 
               
               
                 68 
                 VA0D1_HUMAN 
                 P61421 
                 50% 
                 14 
                 16 
                 2.48 
                 235 
                 
                   3 
                 
               
               
                 69 
                 LPP1_HUMAN 
                 O14494 
                 50% 
                 15 
                 16 
                 4.26 
                 441 
                 
                   3 
                 
               
               
                 70 
                 GP155_HUMAN 
                 Q7Z3F1 
                 50% 
                 1 
                 9 
                 11.64 
                 9 
                 
                   3 
                 
               
               
                 71 
                 1433S_HUMAN 
                 P31947 
                 50% 
                 14 
                 16 
                 
                   0.78 
                 
                 215 
                 
                   3 
                 
               
               
                 72 
                 LRSM1_HUMAN 
                 Q6UWE0 
                 50% 
                 0 
                 8 
                 
                   1.30 
                 
                 15 
                 
                   3 
                 
               
               
                 73 
                 DMA_HUMAN 
                 P28067 
                 50% 
                 7 
                 7 
                 
                   1.67 
                 
                 295 
                 
                   3 
                 
               
               
                 74 
                 RAB9A_HUMAN 
                 P51151 
                 50% 
                 3 
                 9 
                 
                   1.51 
                 
                 68 
                 
                   3 
                 
               
               
                 75 
                 SRC8_HUMAN 
                 Q14247 
                 50% 
                 12 
                 16 
                 
                   0.69 
                 
                 101 
                 
                   3 
                 
               
               
                 76 
                 AQP7_HUMAN 
                 O14520 
                 44% 
                 6 
                 11 
                 2.08 
                 248 
                 
                   3 
                 
               
               
                 77 
                 SYUG_HUMAN 
                 O76070 
                 44% 
                 11 
                 14 
                 2.43 
                 183 
                 
                   3 
                 
               
               
                 78 
                 1433T_HUMAN 
                 P27348 
                 44% 
                 15 
                 16 
                 
                   0.70 
                 
                 175 
                 
                   3 
                 
               
               
                 79 
                 AATC_HUMAN 
                 P17174 
                 44% 
                 15 
                 16 
                 
                   1.55 
                 
                 539 
                 
                   3 
                 
               
               
                 80 
                 CLIC3_HUMAN 
                 O95833 
                 44% 
                 11 
                 13 
                 
                   1.30 
                 
                 156 
                 
                   3 
                 
               
               
                 81 
                 DEST_HUMAN 
                 P60981 
                 44% 
                 15 
                 16 
                 
                   1.49 
                 
                 639 
                 
                   3 
                 
               
               
                 82 
                 RASH_HUMAN 
                 P01112 
                 44% 
                 2 
                 8 
                 
                   1.60 
                 
                 65 
                 
                   3 
                 
               
               
                 83 
                 PTGR2_HUMAN 
                 Q8N8N7 
                 44% 
                 2 
                 7 
                 
                   1.34 
                 
                 94 
                 
                   3 
                 
               
               
                 84 
                 TCPE_HUMAN 
                 P48643 
                 44% 
                 9 
                 14 
                 
                   1.39 
                 
                 13 
                 
                   3 
                 
               
               
                 85 
                 ITIH4_HUMAN 
                 Q14624 
                 40% 
                 6 
                 0 
                 
                   0.23 
                 
                 1 
                 
                   3 
                 
               
               
                 86 
                 AL1A3_HUMAN 
                 P47895 
                 38% 
                 6 
                 14 
                 
                   4.56 
                 
                 97 
                 
                   3 
                 
               
               
                 87 
                 ANXA3_HUMAN 
                 P12429 
                 38% 
                 15 
                 16 
                 
                   2.80 
                 
                 802 
                 
                   3 
                 
               
               
                 88 
                 CLN3_HUMAN 
                 Q13286 
                 38% 
                 1 
                 9 
                 
                   7.26 
                 
                 23 
                 
                   3 
                 
               
               
                 89 
                 CATD_HUMAN 
                 P07339 
                 38% 
                 5 
                 16 
                 
                   4.37 
                 
                 284 
                 
                   3 
                 
               
               
                 90 
                 DDAH1_HUMAN 
                 O94760 
                 38% 
                 15 
                 16 
                 
                   1.84 
                 
                 742 
                 
                   3 
                 
               
               
                 91 
                 NPDC1_HUMAN 
                 Q9NQX5 
                 38% 
                 2 
                 8 
                 
                   4.88 
                 
                 9 
                 
                   3 
                 
               
               
                 92 
                 SAP_HUMAN 
                 P07602 
                 38% 
                 10 
                 14 
                 
                   2.23 
                 
                 115 
                 
                   3 
                 
               
               
                 93 
                 KLK3_HUMAN 
                 P07288 
                 38% 
                 15 
                 16 
                 
                   2.61 
                 
                 783 
                 
                   3 
                 
               
               
                 94 
                 LFG3_HUMAN 
                 Q969X1 
                 38% 
                 15 
                 16 
                 
                   3.32 
                 
                 2745 
                 
                   3 
                 
               
               
                 95 
                 NIBAN_HUMAN 
                 Q9BZQ8 
                 38% 
                 11 
                 16 
                 
                   3.70 
                 
                 210 
                 
                   3 
                 
               
               
                 96 
                 SPNS1_HUMAN 
                 Q9H2V7 
                 38% 
                 1 
                 7 
                 
                   4.38 
                 
                 27 
                 
                   3 
                 
               
               
                 97 
                 LTOR2_HUMAN 
                 Q9Y2Q5 
                 38% 
                 0 
                 6 
                 
                   4.57 
                 
                 226 
                 
                   3 
                 
               
               
                 98 
                 LTOR3_HUMAN 
                 Q9UHA4 
                 38% 
                 0 
                 6 
                 
                   7.91 
                 
                 36 
                 
                   3 
                 
               
               
                 99 
                 SYT7_HUMAN 
                 O43581 
                 38% 
                 7 
                 14 
                 
                   3.90 
                 
                 240 
                 
                   3 
                 
               
               
                 100 
                 T106B_HUMAN 
                 Q9NUM4 
                 38% 
                 0 
                 6 
                 
                   2.53 
                 
                 81 
                 
                   3 
                 
               
               
                 101 
                 MYO5C_HUMAN 
                 Q9NQX4 
                 38% 
                 2 
                 11 
                 
                   15.50 
                 
                 9 
                 
                   3 
                 
               
               
                 102 
                 VAMP2_HUMAN 
                 P63027 
                 38% 
                 14 
                 16 
                 
                   2.23 
                 
                 367 
                 
                   3 
                 
               
               
                 103 
                 VATF_HUMAN 
                 Q16864 
                 38% 
                 7 
                 14 
                 
                   3.18 
                 
                 57 
                 
                   3 
                 
               
               
                 104 
                 AKA7A_HUMAN 
                 O43687 
                 31% 
                 3 
                 13 
                 
                   2.56 
                 
                 24 
                 
                   3 
                 
               
               
                 105 
                 ARSF_HUMAN 
                 P54793 
                 31% 
                 4 
                 13 
                 
                   5.79 
                 
                 75 
                 
                   3 
                 
               
               
                 106 
                 JIP4_HUMAN 
                 O60271 
                 31% 
                 5 
                 12 
                 
                   2.50 
                 
                 34 
                 
                   3 
                 
               
               
                 107 
                 DMBT1_HUMAN 
                 Q9UGM3 
                 31% 
                 7 
                 12 
                 
                   14.32 
                 
                 136 
                 
                   3 
                 
               
               
                 108 
                 GMFB_HUMAN 
                 P60983 
                 31% 
                 1 
                 10 
                 
                   2.52 
                 
                 72 
                 
                   3 
                 
               
               
                 109 
                 FOLH1_HUMAN 
                 Q04609 
                 31% 
                 13 
                 16 
                 
                   2.69 
                 
                 382 
                 
                   3 
                 
               
               
                 110 
                 GSHB_HUMAN 
                 P48637 
                 31% 
                 12 
                 14 
                 
                   3.05 
                 
                 161 
                 
                   3 
                 
               
               
                 111 
                 HPCL1_HUMAN 
                 P37235 
                 31% 
                 13 
                 12 
                 
                   1.79 
                 
                 116 
                 
                   3 
                 
               
               
                 112 
                 MARCS_HUMAN 
                 P29966 
                 31% 
                 15 
                 16 
                 
                   0.38 
                 
                 239 
                 
                   3 
                 
               
               
                 113 
                 NNRE_HUMAN 
                 Q8NCW5 
                 31% 
                 7 
                 12 
                 
                   2.53 
                 
                 226 
                 
                   3 
                 
               
               
                 114 
                 NAPSA_HUMAN 
                 O96009 
                 31% 
                 15 
                 16 
                 
                   2.83 
                 
                 1638 
                 
                   3 
                 
               
               
                 115 
                 AGM1_HUMAN 
                 O95394 
                 31% 
                 8 
                 13 
                 
                   4.08 
                 
                 62 
                 
                   3 
                 
               
               
                 116 
                 AT8A1_HUMAN 
                 Q9Y2Q0 
                 31% 
                 4 
                 12 
                 
                   3.35 
                 
                 50 
                 
                   3 
                 
               
               
                 117 
                 QOR_HUMAN 
                 Q08257 
                 31% 
                 8 
                 14 
                 
                   2.24 
                 
                 175 
                 
                   3 
                 
               
               
                 118 
                 RET5_HUMAN 
                 P82980 
                 31% 
                 14 
                 16 
                 
                   2.36 
                 
                 928 
                 
                   3 
                 
               
               
                 119 
                 SERC2_HUMAN 
                 Q96SA4 
                 31% 
                 15 
                 16 
                 
                   20.18 
                 
                 56 
                 
                   3 
                 
               
               
                 120 
                 S15A1_HUMAN 
                 P46059 
                 31% 
                 0 
                 5 
                 
                   2.23 
                 
                 39 
                 
                   3 
                 
               
               
                 121 
                 SORCN_HUMAN 
                 P30626 
                 31% 
                 14 
                 16 
                 
                   3.22 
                 
                 2828 
                 
                   3 
                 
               
               
                 122 
                 SUIS_HUMAN 
                 P14410 
                 31% 
                 1 
                 5 
                 
                   2.74 
                 
                 4 
                 
                   3 
                 
               
               
                 123 
                 ARPC4_HUMAN 
                 P59998 
                 25% 
                 15 
                 16 
                 
                   2.35 
                 
                 634 
                 
                   3 
                 
               
               
                 124 
                 ARPC5_HUMAN 
                 O15511 
                 25% 
                 15 
                 16 
                 
                   8.06 
                 
                 165 
                 
                   3 
                 
               
               
                 125 
                 APT_HUMAN 
                 P07741 
                 25% 
                 14 
                 16 
                 
                   2.20 
                 
                 356 
                 
                   3 
                 
               
               
                 126 
                 ABRAL_HUMAN 
                 Q9P1F3 
                 25% 
                 5 
                 13 
                 
                   8.38 
                 
                 375 
                 
                   3 
                 
               
               
                 127 
                 IGHA1_HUMAN 
                 P01876 
                 25% 
                 14 
                 16 
                 
                   4.75 
                 
                 593 
                 
                   3 
                 
               
               
                 128 
                 IFM3_HUMAN 
                 Q01628 
                 25% 
                 7 
                 15 
                 
                   10.13 
                 
                 265 
                 
                   3 
                 
               
               
                 129 
                 TRFL_HUMAN 
                 P02788 
                 25% 
                 3 
                 11 
                 
                   41.74 
                 
                 626 
                 
                   3 
                 
               
               
                 130 
                 PNPH_HUMAN 
                 P00491 
                 25% 
                 9 
                 16 
                 
                   2.39 
                 
                 244 
                 
                   3 
                 
               
               
                 131 
                 STXB4_HUMAN 
                 Q6ZWJ1 
                 25% 
                 4 
                 10 
                 
                   2.23 
                 
                 43 
                 
                   3 
                 
               
               
                 132 
                 TSN8_HUMAN 
                 P19075 
                 25% 
                 12 
                 16 
                 
                   0.51 
                 
                 131 
                 
                   3 
                 
               
               
                 133 
                 VPS25_HUMAN 
                 Q9BRG1 
                 25% 
                 14 
                 16 
                 
                   3.06 
                 
                 819 
                 
                   3 
                 
               
               
                 134 
                 VDAC1_HUMAN 
                 P21796 
                 25% 
                 9 
                 16 
                 
                   6.97 
                 
                 116 
                 
                   3 
                 
               
               
                 135 
                 CO6A1_HUMAN 
                 P12109 
                 20% 
                 7 
                 2 
                 
                   0.00 
                 
                 4 
                 
                   3 
                 
               
               
                 136 
                 STAM1_HUMAN 
                 Q92783 
                 20% 
                 12 
                 13 
                 
                   0.63 
                 
                 90 
                 
                   3 
                 
               
               
                 137 
                 CD9_HUMAN 
                 P21926 
                 19% 
                 15 
                 16 
                 
                   2.03 
                 
                 15238 
                 
                   3 
                 
               
               
                 138 
                 FLOT1_HUMAN 
                 O75955 
                 19% 
                 15 
                 16 
                 
                   1.98 
                 
                 589 
                 
                   3 
                 
               
               
                 139 
                 GRAN_HUMAN 
                 P28676 
                 19% 
                 7 
                 7 
                 
                   2.09 
                 
                 723 
                 
                   3 
                 
               
               
                 140 
                 GMPPB_HUMAN 
                 Q9Y5P6 
                 19% 
                 5 
                 14 
                 
                   2.40 
                 
                 71 
                 
                   3 
                 
               
               
                 141 
                 PSA7_HUMAN 
                 O14818 
                 19% 
                 13 
                 13 
                 
                   3.10 
                 
                 244 
                 
                   3 
                 
               
               
                 142 
                 RAB18_HUMAN 
                 Q9NP72 
                 19% 
                 13 
                 16 
                 
                   1.98 
                 
                 243 
                 
                   3 
                 
               
               
                 143 
                 VP37C_HUMAN 
                 A5D8V6 
                 19% 
                 15 
                 16 
                 
                   2.09 
                 
                 761 
                 
                   3 
                 
               
               
                 144 
                 STAM2_HUMAN 
                 O75886 
                 13% 
                 14 
                 11 
                 
                   0.59 
                 
                 51 
                 
                   3 
                 
               
               
                 145 
                 GP2_HUMAN 
                 P55259 
                 13% 
                 4 
                 11 
                 
                   9.07 
                 
                 11 
                 
                   3 
                 
               
               
                 146 
                 PLCG2_HUMAN 
                 P16885 
                  7% 
                 14 
                 11 
                 
                   0.49 
                 
                 40 
                 
                   3 
                 
               
               
                 147 
                 LAMP2_HUMAN 
                 P13473 
                  6% 
                 15 
                 16 
                 
                   2.81 
                 
                 1924 
                 
                   3 
                 
               
               
                 148 
                 CRCT1_HUMAN 
                 Q9UGL9 
                  0% 
                 11 
                 3 
                 
                   0.30 
                 
                 21 
                 
                   3 
                 
               
               
                 149 
                 IPSP_HUMAN 
                 P05154 
                  0% 
                 15 
                 11 
                 
                   0.41 
                 
                 80 
                 
                   3 
                 
               
               
                 150 
                 LCK_HUMAN 
                 P06239 
                  0% 
                 7 
                 2 
                 
                   0.04 
                 
                 35 
                 
                   3 
                 
               
               
                 151 
                 LTOR4 
                 Q0VGL1 
                 — 
                 — 
                 — 
                 
                   7.38 
                 
                 159 
                 
                   3 
                 
               
               
                 152 
                 SCAM1_HUMAN 
                 O15126 
                 56% 
                 2 
                 10 
                 
                   3.88 
                 
                 nd 
                 
                   2 
                 
               
               
                 153 
                 FKB1A_HUMAN 
                 P62942 
                 56% 
                 15 
                 16 
                 1.27 
                 559 
                 
                   2 
                 
               
               
                 154 
                 T176A_HUMAN 
                 Q96HP8 
                 50% 
                 2 
                 10 
                 
                   3.61 
                 
                 nd 
                 
                   2 
                 
               
               
                 155 
                 TYB4_HUMAN 
                 P62328 
                 50% 
                 15 
                 16 
                 1.14 
                 2890 
                 
                   2 
                 
               
               
                 156 
                 HDHD2_HUMAN 
                 Q9H0R4 
                 50% 
                 1 
                 16 
                 1.16 
                 62 
                 
                   2 
                 
               
               
                 157 
                 CDC42_HUMAN 
                 P60953 
                 50% 
                 15 
                 16 
                 1.20 
                 775 
                 
                   2 
                 
               
               
                 158 
                 RAB17_HUMAN 
                 Q9H0T7 
                 50% 
                 10 
                 14 
                 1.23 
                 99 
                 
                   2 
                 
               
               
                 159 
                 CLIC6_HUMAN 
                 Q96NY7 
                 50% 
                 15 
                 16 
                 1.24 
                 217 
                 
                   2 
                 
               
               
                 160 
                 CTL4_HUMAN 
                 Q53GD3 
                 50% 
                 15 
                 16 
                 1.26 
                 1366 
                 
                   2 
                 
               
               
                 161 
                 BLVRB_HUMAN 
                 P30043 
                 50% 
                 15 
                 16 
                 1.27 
                 439 
                 
                   2 
                 
               
               
                 162 
                 RAB10_HUMAN 
                 P61026 
                 50% 
                 15 
                 16 
                 1.41 
                 2078 
                 
                   2 
                 
               
               
                 163 
                 HEBP2_HUMAN 
                 Q9Y5Z4 
                 50% 
                 15 
                 11 
                 1.46 
                 264 
                 
                   2 
                 
               
               
                 164 
                 FABP5_HUMAN 
                 Q01469 
                 50% 
                 9 
                 14 
                 1.53 
                 54 
                 
                   2 
                 
               
               
                 165 
                 SMIM5_HUMAN 
                 Q71RC9 
                 50% 
                 13 
                 13 
                 1.64 
                 3277 
                 
                   2 
                 
               
               
                 166 
                 LAP4A_HUMAN 
                 Q15012 
                 50% 
                 0 
                 8 
                 
                   INF 
                 
                 nd 
                 
                   2 
                 
               
               
                 167 
                 PHYD1_HUMAN 
                 Q5SRE7 
                 50% 
                 0 
                 8 
                 
                   INF 
                 
                 nd 
                 
                   2 
                 
               
               
                 168 
                 PSA5_HUMAN 
                 P28066 
                 50% 
                 0 
                 8 
                 
                   INF 
                 
                 nd 
                 
                   2 
                 
               
               
                 169 
                 CALL3_HUMAN 
                 P27482 
                 44% 
                 4 
                 8 
                 
                   4.79 
                 
                 nd 
                 
                   2 
                 
               
               
                 170 
                 PSN1_HUMAN 
                 P49768 
                 44% 
                 1 
                 7 
                 
                   14.95 
                 
                 nd 
                 
                   2 
                 
               
               
                 171 
                 NQO2_HUMAN 
                 P16083 
                 44% 
                 5 
                 13 
                 1.03 
                 70 
                 
                   2 
                 
               
               
                 172 
                 TCTP_HUMAN 
                 P13693 
                 44% 
                 10 
                 16 
                 1.20 
                 244 
                 
                   2 
                 
               
               
                 173 
                 LAMP1_HUMAN 
                 P11279 
                 44% 
                 15 
                 16 
                 1.38 
                 1279 
                 
                   2 
                 
               
               
                 174 
                 CD38_HUMAN 
                 P28907 
                 44% 
                 7 
                 13 
                 1.53 
                 28 
                 
                   2 
                 
               
               
                 175 
                 MTPN_HUMAN 
                 P58546 
                 44% 
                 15 
                 16 
                 
                   INF 
                 
                 nd 
                 
                   2 
                 
               
               
                 176 
                 DYL2_HUMAN 
                 Q96FJ2 
                 38% 
                 10 
                 14 
                 1.95 
                 34 
                 
                   2 
                 
               
               
                 177 
                 DHPR_HUMAN 
                 P09417 
                 38% 
                 15 
                 16 
                 2.02 
                 122 
                 
                   2 
                 
               
               
                 178 
                 NADC_HUMAN 
                 Q15274 
                 38% 
                 4 
                 10 
                 4.34 
                 21 
                 
                   2 
                 
               
               
                 179 
                 CC50A_HUMAN 
                 Q9NV96 
                 38% 
                 8 
                 10 
                 
                   1.26 
                 
                 48 
                 
                   2 
                 
               
               
                 180 
                 PMVK_HUMAN 
                 Q15126 
                 38% 
                 5 
                 8 
                 
                   0.70 
                 
                 16 
                 
                   2 
                 
               
               
                 181 
                 IF4H_HUMAN 
                 Q15056 
                 31% 
                 11 
                 15 
                 1.86 
                 155 
                 
                   2 
                 
               
               
                 182 
                 TTYH3_HUMAN 
                 Q9C0H2 
                 31% 
                 15 
                 16 
                 2.11 
                 132 
                 
                   2 
                 
               
               
                 183 
                 NPT2B_HUMAN 
                 O95436 
                 31% 
                 2 
                 11 
                 2.72 
                 9 
                 
                   2 
                 
               
               
                 184 
                 LAC2_HUMAN(+1) 
                 P0CG05 
                 31% 
                 4 
                 9 
                 3.28 
                 31 
                 
                   2 
                 
               
               
                 185 
                 RABP2_HUMAN 
                 P29373 
                 31% 
                 15 
                 15 
                 3.63 
                 389 
                 
                   2 
                 
               
               
                 186 
                 CUTA_HUMAN 
                 O60888 
                 31% 
                 6 
                 12 
                 4.03 
                 104 
                 
                   2 
                 
               
               
                 187 
                 PSA4_HUMAN 
                 P25789 
                 31% 
                 4 
                 10 
                 6.11 
                 13 
                 
                   2 
                 
               
               
                 188 
                 S35F6_HUMAN 
                 Q8N357 
                 31% 
                 3 
                 11 
                 6.24 
                 25 
                 
                   2 
                 
               
               
                 189 
                 HEM2_HUMAN 
                 P13716 
                 31% 
                 10 
                 14 
                 
                   1.73 
                 
                 63 
                 
                   2 
                 
               
               
                 190 
                 DCXR_HUMAN 
                 Q7Z4W1 
                 31% 
                 15 
                 16 
                 
                   1.58 
                 
                 496 
                 
                   2 
                 
               
               
                 191 
                 UPK1A_HUMAN 
                 O00322 
                 31% 
                 15 
                 16 
                 
                   1.53 
                 
                 3427 
                 
                   2 
                 
               
               
                 192 
                 SPR1A_HUMAN 
                 P35321 
                 25% 
                 8 
                 14 
                 2.64 
                 177 
                 
                   2 
                 
               
               
                 193 
                 ZN185_HUMAN 
                 O15231 
                 25% 
                 5 
                 10 
                 4.49 
                 1 
                 
                   2 
                 
               
               
                 194 
                 TMM8A_HUMAN 
                 Q9HCN3 
                 25% 
                 1 
                 7 
                 6.47 
                 6 
                 
                   2 
                 
               
               
                 195 
                 PCYOX_HUMAN 
                 Q9UHG3 
                 25% 
                 1 
                 13 
                 8.22 
                 24 
                 
                   2 
                 
               
               
                 196 
                 LYSC_HUMAN 
                 P61626 
                 25% 
                 10 
                 16 
                 23.11 
                 1449 
                 
                   2 
                 
               
               
                 197 
                 PALM_HUMAN 
                 O75781 
                 19% 
                 2 
                 9 
                 3.70 
                 3 
                 
                   2 
                 
               
               
                 198 
                 CEAM6_HUMAN 
                 P40199 
                 19% 
                 5 
                 8 
                 5.05 
                 352 
                 
                   2 
                 
               
               
                 199 
                 SC5A1_HUMAN 
                 P13866 
                 19% 
                 15 
                 16 
                 
                   1.75 
                 
                 472 
                 
                   2 
                 
               
               
                 200 
                 PTGR1_HUMAN 
                 Q14914 
                 13% 
                 15 
                 16 
                 
                   1.57 
                 
                 1125 
                 
                   2 
                 
               
               
                 201 
                 S10A9_HUMAN 
                 P06702 
                 13% 
                 10 
                 14 
                 
                   1.31 
                 
                 1928 
                 
                   2 
                 
               
               
                 202 
                 MILK1_HUMAN 
                 Q8N3F8 
                  6% 
                 4 
                 6 
                 3.41 
                 1 
                 
                   2 
                 
               
               
                 203 
                 ABHEB_HUMAN 
                 Q96IU4 
                  6% 
                 14 
                 16 
                 
                   0.93 
                 
                 652 
                 
                   2 
                 
               
               
                 204 
                 AQP2_HUMAN 
                 P41181 
                  6% 
                 15 
                 16 
                 
                   1.59 
                 
                 6762 
                 
                   2 
                 
               
               
                 205 
                 GSTP1_HUMAN 
                 P09211 
                  6% 
                 15 
                 16 
                 
                   1.71 
                 
                 2655 
                 
                   2 
                 
               
               
                 206 
                 ZDHC1_HUMAN 
                 Q8WTX9 
                  0% 
                 15 
                 15 
                 0.57 
                 175 
                 
                   2 
                 
               
               
                 207 
                 RAB8B_HUMAN 
                 Q92930 
                 56% 
                 14 
                 16 
                 
                   1.30 
                 
                 nd 
                 
                   1 
                 
               
               
                 208 
                 TMPS2_HUMAN 
                 O15393 
                 44% 
                 15 
                 16 
                 
                   1.16 
                 
                 nd 
                 
                   1 
                 
               
               
                 209 
                 RAB1B_HUMAN 
                 Q9H0U4 
                 44% 
                 15 
                 16 
                 
                   1.35 
                 
                 nd 
                 
                   1 
                 
               
               
                 210 
                 RAB1A_HUMAN 
                 P62820 
                 44% 
                 15 
                 16 
                 
                   1.47 
                 
                 nd 
                 
                   1 
                 
               
               
                 211 
                 RAB43_HUMAN 
                 Q86YS6 
                 38% 
                 2 
                 8 
                 
                   7.56 
                 
                 nd 
                 
                   1 
                 
               
               
                 212 
                 SNG2_HUMAN 
                 O43760 
                 38% 
                 1 
                 6 
                 
                   8.43 
                 
                 nd 
                 
                   1 
                 
               
               
                 213 
                 2B1F_HUMAN 
                 P01911 
                 38% 
                 1 
                 10 
                 
                   9.13 
                 
                 nd 
                 
                   1 
                 
               
               
                 214 
                 NCUG1_HUMAN 
                 Q8WWB7 
                 38% 
                 1 
                 6 
                 
                   23.59 
                 
                 nd 
                 
                   1 
                 
               
               
                 215 
                 CAB39_HUMAN 
                 Q9Y376 
                 38% 
                 15 
                 16 
                 0.89 
                 1074 
                 
                   1 
                 
               
               
                 216 
                 DYN2_HUMAN 
                 P50570 
                 38% 
                 13 
                 16 
                 0.98 
                 96 
                 
                   1 
                 
               
               
                 217 
                 C42S2_HUMAN 
                 Q9NRR3 
                 38% 
                 8 
                 14 
                 1.20 
                 775 
                 
                   1 
                 
               
               
                 218 
                 FRIH_HUMAN 
                 P02794 
                 38% 
                 11 
                 14 
                 1.61 
                 655 
                 
                   1 
                 
               
               
                 219 
                 S35F2_HUMAN 
                 Q8IXU6 
                 38% 
                 0 
                 6 
                 
                   INF 
                 
                 nd 
                 
                   1 
                 
               
               
                 220 
                 PNKD_HUMAN 
                 Q8N490 
                 33% 
                 10 
                 6 
                 1.52 
                 9 
                 
                   1 
                 
               
               
                 221 
                 CATZ_HUMAN 
                 Q9UBR2 
                 31% 
                 5 
                 10 
                 
                   2.52 
                 
                 nd 
                 
                   1 
                 
               
               
                 222 
                 TBB2B_HUMAN 
                 Q9BVA1 
                 31% 
                 2 
                 9 
                 
                   7.48 
                 
                 nd 
                 
                   1 
                 
               
               
                 223 
                 TSTD1_HUMAN 
                 Q8NFU3 
                 31% 
                 11 
                 15 
                 0.93 
                 132 
                 
                   1 
                 
               
               
                 224 
                 MK01_HUMAN 
                 P28482 
                 31% 
                 9 
                 16 
                 1.01 
                 54 
                 
                   1 
                 
               
               
                 225 
                 ADHX_HUMAN 
                 P11766 
                 31% 
                 11 
                 16 
                 1.12 
                 178 
                 
                   1 
                 
               
               
                 226 
                 PPAC_HUMAN 
                 P24666 
                 31% 
                 7 
                 15 
                 1.31 
                 52 
                 
                   1 
                 
               
               
                 227 
                 ANXA4_HUMAN 
                 P09525 
                 31% 
                 15 
                 16 
                 1.36 
                 1971 
                 
                   1 
                 
               
               
                 228 
                 SEPT2_HUMAN 
                 Q15019 
                 31% 
                 14 
                 16 
                 1.44 
                 92 
                 
                   1 
                 
               
               
                 229 
                 GSTM3_HUMAN 
                 P21266 
                 31% 
                 15 
                 16 
                 1.46 
                 781 
                 
                   1 
                 
               
               
                 230 
                 BRK1_HUMAN 
                 Q8WUW1 
                 31% 
                 1 
                 8 
                 1.48 
                 41 
                 
                   1 
                 
               
               
                 231 
                 PSB2_HUMAN 
                 P49721 
                 31% 
                 0 
                 5 
                 
                   INF 
                 
                 nd 
                 
                   1 
                 
               
               
                 232 
                 UBE2K_HUMAN 
                 P61086 
                 31% 
                 0 
                 5 
                 
                   INF 
                 
                 nd 
                 
                   1 
                 
               
               
                 233 
                 S10A1_HUMAN 
                 P23297 
                 25% 
                 6 
                 11 
                 
                   2.46 
                 
                 nd 
                 
                   1 
                 
               
               
                 234 
                 MAP1A_HUMAN 
                 P78559 
                 25% 
                 1 
                 7 
                 
                   4.87 
                 
                 nd 
                 
                   1 
                 
               
               
                 235 
                 GSTM1_HUMAN 
                 P09488 
                 25% 
                 10 
                 14 
                 1.34 
                 381 
                 
                   1 
                 
               
               
                 236 
                 MMP24_HUMAN 
                 Q9Y5R2 
                 20% 
                 14 
                 5 
                 
                   0.39 
                 
                 nd 
                 
                   1 
                 
               
               
                 237 
                 SIM22_HUMAN 
                 K7EJ46 
                 19% 
                 10 
                 14 
                 
                   2.05 
                 
                 nd 
                 
                   1 
                 
               
               
                 238 
                 HGNAT_HUMAN 
                 Q68CP4 
                 19% 
                 3 
                 16 
                 
                   4.28 
                 
                 nd 
                 
                   1 
                 
               
               
                 239 
                 SARG_HUMAN 
                 Q9BW04 
                 19% 
                 15 
                 16 
                 1.06 
                 335 
                 
                   1 
                 
               
               
                 240 
                 ABI1_HUMAN 
                 Q8IZP0 
                 19% 
                 15 
                 16 
                 1.09 
                 190 
                 
                   1 
                 
               
               
                 241 
                 CF132_HUMAN 
                 Q5T0Z8 
                 19% 
                 4 
                 8 
                 1.16 
                 8 
                 
                   1 
                 
               
               
                 242 
                 ARF5_HUMAN 
                 P84085 
                 13% 
                 6 
                 12 
                 0.91 
                 56 
                 
                   1 
                 
               
               
                 243 
                 IDHC_HUMAN 
                 O75874 
                 13% 
                 15 
                 16 
                 1.22 
                 2420 
                 
                   1 
                 
               
               
                 244 
                 GLTP_HUMAN 
                 Q9NZD2 
                 13% 
                 14 
                 10 
                 1.48 
                 167 
                 
                   1 
                 
               
               
                 245 
                 TPM4_HUMAN 
                 P67936 
                  6% 
                 9 
                 14 
                 
                   2.47 
                 
                 nd 
                 
                   1 
                 
               
               
                 246 
                 NRAM2_HUMAN 
                 P49281 
                  6% 
                 10 
                 16 
                 
                   1.63 
                 
                 nd 
                 
                   0 
                 
               
               
                   
               
               
                 *CD59 found in all samples, but only in CTR in validation analysis 
               
               
                 **Lactotransferrin found exclusively in fresh (2nd) urine 
               
               
                 ***LAMTOR4 only found validation analysis, not in discovery analysis 
               
               
                 Column: Validated iBAQ ratio PAT:CTR 
               
               
                 Bold = found significantly altered (p &lt; 0.05) in PAT in validation analysis 
               
               
                 Normal = found in validation analysis (p &gt; 0.05), (iBAQ ratio) 
               
               
                 Italics = not found in validation analysis, values from discovery analysis (TOP3TIC ratio) 
               
               
                 INF = Infinite; only found in PAT samples 
               
               
                 Ratios of &lt;1 indicate that those proteins have a reduced level in urinary exosomes from prostate cancer samples compared to a control level. 
               
               
                 Column: Abundance 
               
               
                 Abundance: for PAT:CTR &gt; 1.0; total detected protein amount in PAT, for PAT:CTR &lt; 1.0; total detected protein amount in CTR 
               
               
                 nd = not detected 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Individual patient sample diagnostic calls associated with the most promising markers. A positive 
               
               
                 diagnostic call (expression above diagnostic threshold at 100% specificity) is indicated by ‘1’. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Sequence 
                 Sensitivity 
                 P1 
                 P2 
                 P3 
                 P4 
                 P5 
                 P6 
                 P7 
                 P8 
                 P9 
                 P10 
                 P12 
                 P13 
                 P14 
                 P15 
                 P16 
                 P17 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 94% 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 2 
                 81% 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 3 
                 81% 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
               
               
                 4 
                 75% 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 5 
                 75% 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 6 
                 75% 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 7 
                 75% 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 8 
                 75% 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 9 
                 75% 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
               
               
                 10 
                 69% 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
               
               
                 11 
                 69% 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 12 
                 69% 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 13 
                 69% 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
               
               
                 14 
                 63% 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
               
               
                 15 
                 63% 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 0 
                 1 
               
               
                 16 
                 63% 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 17 
                 63% 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 18 
                 56% 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
               
               
                 19 
                 56% 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
               
               
                 20 
                 56% 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
               
               
                 21 
                 56% 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 0 
                 0 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
               
               
                 22 
                 56% 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 23 
                 56% 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
               
               
                 24 
                 56% 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 25 
                 56% 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 26 
                 56% 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 0 
                 1 
                 1 
                 0 
               
               
                 27 
                 56% 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 1 
               
               
                 28 
                 50% 
                 1 
                 1 
                 0 
                 1 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
               
               
                 29 
                 50% 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 30 
                 50% 
                 0 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
               
               
                 31 
                 50% 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 32 
                 50% 
                 1 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
               
               
                 33 
                 50% 
                 1 
                 1 
                 1 
                 0 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 34 
                 50% 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 35 
                 50% 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
               
               
                 36 
                 50% 
                 1 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
               
               
                 37 
                 50% 
                 1 
                 0 
                 1 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 38 
                 44% 
                 1 
                 1 
                 0 
                 1 
                 0 
                 1 
                 0 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
               
               
                 39 
                 44% 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 40 
                 44% 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 41 
                 44% 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 1 
               
               
                 42 
                 44% 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 43 
                 44% 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 1 
               
               
                 44 
                 44% 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
               
               
                 45 
                 44% 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 46 
                 44% 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
               
               
                 47 
                 44% 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
               
               
                 48 
                 44% 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 49 
                 44% 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 50 
                 44% 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 51 
                 44% 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 52 
                 44% 
                 1 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
               
               
                 53 
                 44% 
                 0 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
               
               
                 54 
                 44% 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
               
               
                 55 
                 44% 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
               
               
                 56 
                 44% 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
               
               
                 57 
                 44% 
                 0 
                 1 
                 0 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
               
               
                 58 
                 41% 
                 1 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Differentially expressed proteins from urinary exosomes which  
               
               
                 were also previously identified in exosomes of the PC-3 cell line.  
               
               
                 Proteins in bold are included in the focus list of Table 3. 
               
            
           
           
               
               
               
            
               
                   
                 Uniprot 
                   
               
               
                   
                 Ace. No. 
                 Full Protein Name 
               
               
                   
                   
               
               
                   
                 P31947 
                 14-3-3 protein sigma 
               
               
                   
                 P27348 
                 14-3-3 protein theta 
               
               
                   
                 P59998 
                 Actin-related protein 2/3 complex subunit 4 
               
               
                   
                 O15511 
                 Actin-related protein 2/3 complex subunit 5 
               
               
                   
                 
                   Q9NVJ2 
                 
                 
                   ADP-ribosylation 
                   factor-like 
                   protein 
                   8B 
                 
               
               
                   
                 P12429 
                 Annexin A3 
               
               
                   
                 P61769 
                 Beta-2-microglobulin 
               
               
                   
                 Q9Y376 
                 Calcium-binding protein 39 
               
               
                   
                 
                   P62158 
                 
                 
                   Calmodulin 
                 
               
               
                   
                 
                   P60033 
                 
                 
                   CD81 
                   antigen 
                 
               
               
                   
                 P21926 
                 CD9 antigen 
               
               
                   
                 
                   O15551 
                 
                 
                   Claudin-3 
                 
               
               
                   
                 P60981 
                 Destrin 
               
               
                   
                 P02794 
                 Ferritin heavy chain 
               
               
                   
                 O75955 
                 Flotillin-1 
               
               
                   
                 P29966 
                 Myristoylated alanine-rich C-kinase substrate 
               
               
                   
                 
                   P13796 
                 
                 
                   Plastin-2 
                 
               
               
                   
                 
                   Q99497 
                 
                 
                   Protein 
                   DJ-1 
                 
               
               
                   
                 P61026 
                 Ras-related protein Rab-10 
               
               
                   
                 Q6IQ22 
                 Ras-related protein Rab-12 
               
               
                   
                 P61106 
                 Ras-related protein Rab-14 
               
               
                   
                 P62820 
                 Ras-related protein Rab-1A 
               
               
                   
                 Q9H0U4 
                 Ras-related protein Rab-1B 
               
               
                   
                 
                   P51149 
                 
                 
                   Ras-related 
                   protein 
                   Rab-7a 
                 
               
               
                   
                 P61006 
                 Ras-related protein Rab-8A 
               
               
                   
                 Q92930 
                 Ras-related protein Rab-8B 
               
               
                   
                 Q15019 
                 Septin-2 
               
               
                   
                 P13693 
                 Translationally-controlled tumor protein 
               
               
                   
                 P63027 
                 Vesicle-associated membrane protein 2 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Examples of biomarker multiplexing for improved diagnostic accuracy. A positive diagnostic 
               
               
                 call (expression above diagnostic threshold at 100% specificity) is indicated by ‘1’. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Sequence 
                 P1 
                 P2 
                 P3 
                 P4 
                 P5 
                 P6 
                 P7 
                 P8 
                 P9 
                 P10 
                 P12 
                 P13 
                 P14 
                 P15 
                 P16 
                 P17 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 3 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
               
               
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 9 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
               
               
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 13 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
               
               
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 20 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
               
               
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 35 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
               
               
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 56 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
               
               
                 2 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 3 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
               
               
                 3 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
               
               
                 5 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 3 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
               
               
                 6 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 3 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
               
               
                 8 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 3 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
               
               
                 11 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 3 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
               
               
                 13 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
               
               
                 3 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
               
               
                 17 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 9 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
               
               
                 6 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 9 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
               
               
                 7 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 9 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
               
               
                 11 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 9 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
               
               
                 12 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 13 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
               
               
                 15 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 0 
                 1 
               
               
                 3 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
               
               
                 4 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 10 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
               
               
                 3 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
               
               
                 14 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 0 
                 1 
               
               
                 16 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 3 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
               
               
                 10 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
               
               
                 12 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 0 
               
               
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
               
               
                 2 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 1 
               
               
                 3 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
               
               
                 9 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 0 
                 1 
                 1 
                 1 
                 0 
                 1 
                 0 
                 0 
                 1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Diagnostic properties of markers at diagnostic thresholds set to  
               
               
                 optimize the combination of sensitivity and specificity 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEN +  
               
               
                 Full protein name 
                 SEN 
                 SPE 
                 SPE 
               
               
                   
               
               
                 Transmembrane protein 256 
                 100% 
                  94% 
                 194% 
               
               
                 Adipogenesis regulatory factor 
                 100% 
                  81% 
                 181% 
               
               
                 Ragulator complex protein LAMTOR1 
                  87% 
                  94% 
                 180% 
               
               
                 
                   Vesicle-associated 
                   membrane 
                   protein 
                   2 
                 
                   80%   
                 
                   100% 
                 
                 
                   180% 
                 
               
               
                 V-type proton ATPase 16 kDa proteolipid subunit 
                 100% 
                  75% 
                 175% 
               
               
                 Acid ceramidase 
                  93% 
                  81% 
                 175% 
               
               
                 
                   Prenylcysteine 
                   oxidase 
                   1 
                 
                   93%   
                   81%   
                 
                   175% 
                 
               
               
                 
                   Sorcin 
                 
                   87%   
                   88%   
                 
                   174% 
                 
               
               
                 
                   Grancalcin 
                 
                   80%   
                   94%   
                 
                   174% 
                 
               
               
                 Ras-related protein Rab-7a 
                  80% 
                  94% 
                 174% 
               
               
                 Tetraspanin-6 
                  80% 
                  94% 
                 174% 
               
               
                 3-hydroxybutyrate dehydrogenase type 2 
                  87% 
                  81% 
                 168% 
               
               
                 EF-hand domain-containing protein D2 
                  87% 
                  81% 
                 168% 
               
               
                 Flotillin-2 
                  87% 
                  81% 
                 168% 
               
               
                 Ras-related protein Rab-3D 
                  80% 
                  88% 
                 168% 
               
               
                 Adenine phosphoribosyltransferase 
                  73% 
                  94% 
                 167% 
               
               
                 Calmodulin 
                  73% 
                  94% 
                 167% 
               
               
                 Protein DJ-1 
                  73% 
                  94% 
                 167% 
               
               
                 Retinol-binding protein 5 
                  73% 
                  94% 
                 167% 
               
               
                 Ubiquitin-conjugating enzyme E2 variant 2 
                  73% 
                  94% 
                 167% 
               
               
                 S-phase kinase-associated protein 1 
                  67% 
                 100% 
                 167% 
               
               
                 ADP-ribosylation factor-like protein 8B 
                  93% 
                  69% 
                 162% 
               
               
                 Cytochrome b561 
                  93% 
                  69% 
                 162% 
               
               
                 GDP-mannose 4.6 dehydratase 
                  93% 
                  69% 
                 162% 
               
               
                 Matrix metalloproteinase-24 
                  69% 
                  93% 
                 162% 
               
               
                 CD59 glycoprotein 
                  87% 
                  75% 
                 162% 
               
               
                 Claudin-2 
                  87% 
                  75% 
                 162% 
               
               
                 Glutathione synthetase 
                  87% 
                  75% 
                 162% 
               
               
                 Costars family protein ABRACL 
                  80% 
                  81% 
                 161% 
               
               
                 Low molecular weight phosphotyrosine protein 
                  80% 
                  81% 
                 161% 
               
               
                 A-kinase anchor protein 7 isoforms alpha and beta 
                  80% 
                  81% 
                 161% 
               
               
                 Actin-related protein 2/3 complex subunit 4 
                  80% 
                  81% 
                 161% 
               
               
                 2′-deoxynucleoside 5′-phosphate N-hydrolase 1  
                  80% 
                  81% 
                 161% 
               
               
                 Dihydropteridine reductase 
                  80% 
                  81% 
                 161% 
               
               
                 Ras-related protein Rab-3B 
                  80% 
                  81% 
                 161% 
               
               
                 Vacuolar protein-sorting-associated protein 25 
                  80% 
                  81% 
                 161% 
               
               
                 Cathepsin D 
                  73% 
                  88% 
                 161% 
               
               
                 L-xylulose reductase 
                  73% 
                  88% 
                 161% 
               
               
                 Mannose-1-phosphate guanyltransferase beta 
                  73% 
                  88% 
                 161% 
               
               
                 Napsin-A 
                  73% 
                  88% 
                 161% 
               
               
                 Purine nucleoside phosphorylase 
                  73% 
                  88% 
                 161% 
               
               
                 Prostaglandin reductase 1 
                  73% 
                  88% 
                 161% 
               
               
                 Ras-related protein Rab-2A 
                  73% 
                  88% 
                 161% 
               
               
                 Protein S100-P 
                  73% 
                  88% 
                 161% 
               
               
                 Small integral membrane protein 22 
                  73% 
                  88% 
                 161% 
               
               
                 Synaptotagmin-7 
                  73% 
                  88% 
                 161% 
               
               
                 Tetraspanin-8 
                  73% 
                  88% 
                 161% 
               
               
                 Annexin A4 
                  67% 
                  94% 
                 160% 
               
               
                 CD81 antigen 
                  67% 
                  94% 
                 160% 
               
               
                 Flotillin-1 
                  67% 
                  94% 
                 160% 
               
               
                 Interferon-induced transmembrane protein 3 
                  67% 
                  94% 
                 160% 
               
               
                 Nicastrin 
                  67% 
                  94% 
                 160% 
               
               
                 Lipid phosphate phosphohydrolase 1 
                  67% 
                  94% 
                 160% 
               
               
                 Ras-related protein Rab-18 
                  67% 
                  94% 
                 160% 
               
               
                 Transmembrane protein 63A 
                  67% 
                  94% 
                 160% 
               
               
                 Transmembrane protease serine 2 
                  67% 
                  94% 
                 160% 
               
               
                 UDP-glucose 6-dehydrogenase 
                  67% 
                  94% 
                 160% 
               
               
                   
               
            
           
         
       
     
     Example 2 
     Introduction 
     In Example 1, we identified 246 proteins differentially expressed in urinary exosomes from prostate cancer patients (16) compared to normal individuals (15) by mass spectrometry (MS). From this analysis, we defined a short list of the most diagnostically promising proteins, demonstrating high individual sensitivity and specificity for prostate cancer. 
     MS is not yet widely used in clinical laboratories. We have thus investigated the possibility to transfer the identified biomarkers to an immunoassay based analysis platform, which would better integrate into current clinical lab routines. We have obtained commercially available antibodies and ELISA assays for some of the candidate biomarkers. These have been tested in biological samples, and employed to demonstrate the feasibility to transfer the MS-identified biomarkers to an immunoassay platform. 
     Materials and Methods 
     Materials 
     ProteoSilver Plus Silver Stain kit was purchased from Sigma-Aldrich (St. Louis, Mo., USA). Bicinchoninic acid (BCA) protein assay kit was from Pierce (Thermo Scientific, Rockford, Ill., USA). Mini-protean TGX gels and Tranfer-Blot Turbo Transfer Pack were from Bio-Rad (Hercules, Calif., USA). The primary antibodies used for Western blotting were: mouse anti-Flotillin 1 (BD Biosciences), mouse anti-mouse flotillin 2 (BD Biosciences), rabbit anti-Rab3B (Abcam), rabbit anti- LAMTOR1 (Abcam), rabbit anti-TMEM256 (Abcam). HRP-conjugated secondary antibodies were from Jackson Immunoresearch (West Grove, Pa., USA). The DJ-1/PARK? ELISA Kit (CY-9050V2) was from MBL and the Flotillin 2 ELISA kit (ABIN418175) was from Antibodies-online.com 
     Urine Collection and Exosome Isolation 
     Urine collection and exosome isolation was performed as described in Example 1 and published in Øverbye A. et al, 2015, Oncotarget. 6(30):30357-76. 
     Total Protein Quantification 
     The amount of total protein in exosomes was determined using a BCA assay kit according to the manufacturer&#39;s instructions. BSA was used as standard protein. 
     SDS-PAGE and Silver Staining 
     Similar amounts of urinary exosomes were mixed with loading buffer, and the samples were run on 4-20% polyacrylamide gels. The gels were stained using ProteoSilver Plus Silver Stain kit following the manufacturer&#39;s protocol. 
     SDS-PAGE and Immunoblotting 
     Similar amounts of urinary exosomes were solubilised in loading buffer and run on 4-20% gradient TGX gels. The proteins were transferred to PVDF membranes using a Tranfer-Blot Turbo Transfer Pack. Membranes were incubated with the specified primary and secondary antibodies. Blots were visualized with the Amersham™ ECL™ Prime Western blot detection (GE Healthcare, Little Chalfont, UK) on the Universal Hood II Bio-Rad scanner (Bio-Rad, Hercules, Calif., USA). 
     ELISA Assays 
     Similar amounts of urinary exosomes were analyzed following the manufacturer&#39;s protocol. 
     Results 
     Urine was collected and exosomes isolated as previously described in Example 1 and Øverbye A. et al, 2015, Oncotarget 6(30):30357-76. In order to analyze similar amounts of urinary exosomes from the different individuals, the protein amount of exosomes was measured by the BCA assay and/or by the intensity of silver stained samples (data not shown). First, Western blot experiments designed to detect flotillin1, flotillin2, TM256, Rab-3B and LAMTOR1 were performed. In order to identify the amount of exosomes required to detect specific proteins by Western blot, several amounts of exosomes were loaded on gels. As shown in  FIG. 3A , relatively low amounts of exosomes were required in order to detect these proteins by Western blot. The intensity of the bands was related to the sample amount/concentration to produce a standard curve as shown in  FIG. 3B  for flotillin2. Then, similar protein amounts of urinary exosomes from 9 healthy controls and 9 patients were analyzed. In agreement with the mass spectrometry data (Example 1, Øverbye A. et al, 2015, Oncotarget 6(30):30357-76), there were clear differences in the levels of the tested proteins between healthy control males and prostate cancer patients. The patient to control ratio for the different proteins (based on the sum of the individual intensities for each protein in the control and in the patient group) is shown in  FIG. 4 . Furthermore, analysis of individual Western blot signals for flotillin2 in 16 controls and 16 patients showed 87.5% sensitivity (14/16 patients corrected identified as positive) and 94% specificity (15/16 controls correctly identified as negative) at an optimally set expression threshold ( FIG. 5 ). 
     As a next step, ELISA assays were performed to validate the Western blot results of two protein markers, flotillin 2 and PARK7 (Protein DJ-1). Since flotillin2 is expected to be located in the exosomal lumen, exosomes solubilized in 0.5% Triton X-100 were used in these experiments. Control experiments showed that the ELISA kit was compatible with this concentration of Triton X-100. Standard curves were created for both protein markers and different amounts of control urinary exosomes were tested (data not shown). Once the amount of urinary exosomes required to detect the proteins with the ELISA kit were calculated, similar amounts of control and patient samples were analyzed. The ELISA assays indicate that levels for both proteins were higher in the prostate cancer samples than in healthy controls (1.5 fold higher for flotillin2, 1.8 fold higher for PARK7), in general agreement with the Western blot and the MS data. 
     In conclusion, our data support the feasibility of detecting the MS-identified protein biomarkers in urinary exosomes by antibody based detection methods.