Patent Publication Number: US-2023139188-A1

Title: Markers for renal disease

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional application of U.S. application Ser. No. 16/571,345, filed on Sep. 16, 2019, which is a divisional of U.S. application Ser. No. 13/700,992, filed on Jan. 30, 2013, now U.S. Pat. No. 10,436,797, issued Oct. 8, 2019, which is a U.S. National Stage Application of PCT/US2011/039122, filed on Jun. 3, 2011, which claims the benefit of U.S. Provisional Application Nos. 61/351,183, filed Jun. 3, 2010, and 61/411,280 filed Nov. 8, 2010, all of which are incorporated herein by reference in their entireties. 
    
    
     SEQUENCE LISTING 
     This document incorporates by reference an electronic sequence listing text file. The text file is named 10712WOUSSL.txt, is 14.0 kilobytes, and was created on Jan. 29, 2013. 
     BACKGROUND OF THE INVENTION 
     Renal disease is associated with increased water consumption, frequent urination, diminished appetite, weight loss and muscle atrophy. Generally, by the time clinical symptoms of renal disease develop, irreparable kidney damage has occurred. Early detection permits earlier treatment and in turn slows disease progression. Current treatment includes dialysis and a diet low in phosphorous and protein. Unfortunately, no cure for chronic renal disease exists and kidney failure will eventually occur. Therefore, early detection is crucial for improved life span and quality of life. 
     In mammals, renal disease progression is divided into five levels. Current methods for detecting canine renal disease include kidney ultrasound, biopsy, or measurement of urine protein/creatinine levels. Biopsy is invasive and creatinine measurement is not accurate until stage three of renal failure, which is after significant tissue damage has occurred. Methods for detecting canine renal disease at earlier stages are needed in the art as such methods would inhibit disease progression. 
     SUMMARY OF THE INVENTION 
     This invention provides reagents and methods for identifying patients with renal disease. The reagents and methods of this invention are directed to detecting levels of specific metabolites, full-length proteins and protein fragments, particularly inosine nucleoside and the following proteins: apolipoprotein C-I, apolipoprotein C-II, fibrinogen alpha chain, or fibrinogen A-alpha chain, kininogen, keratin Type I cytoskeletol 10, cystatin A, cystatin B, Inter-Alpha Inhibitor H4 (ITIH4) and/or one or more of SEQ ID NOs:1-59 in renal patient samples. The relative levels of full-length protein and protein fragment provide biomarkers for diagnosing kidney/renal disease. Reagents and methods of this invention are additionally directed to assessing inosine concentrations as a biomarker for kidney/renal disease. Specific embodiments of the reagents and methods of the described invention are adapted for detecting protein biomarkers specific to renal disease. In one embodiment, antibodies specific for SEQ ID NOS: 3, 7, 13, or 20 are used to bind proteins and protein fragments produced in patients with renal disease; a non-limiting example of such proteins identified herein include apolipoprotein C-I, apolipoprotein C-II, fibrinogen alpha chain, or fibrinogen A-alpha chain. In a further embodiment, antibodies are specific for CysB1, Cys A, Kininogen, Inter-Alpha Inhibitor H4 (ITIH4), or keratin type I cytoskeletal 10. In a particular embodiment, methods for assessing the differential levels of inosine provide a biomarker for renal disease. Inosine levels may be assessed, for example, by LC/MS or inosine-specific antibodies. In additional embodiments, the reagents and methods provided herein detect altered protein levels in blood, serum, plasma, or urine. A plurality of altered protein and protein fragments are disclosed herein that occur in renal disease, including but not limited to amino acid sequences set forth in greater detail (see Table 1). Certain embodiments of the invention also provide one or a plurality of polypeptide sequences disclosed herein that exhibit altered levels in renal patient samples. In additional embodiments, the invention provides diagnostic methods using antibodies specific to one or a plurality of polypeptides consisting of SEQ ID NOS: 1-59 for identifying renal disease. 
     An embodiment of the invention provides antibodies that specifically bind to one or a plurality of polypeptides consisting of SEQ ID NOS: 1-59. In a preferred embodiment, the invention provides an antibody that specifically binds to a polypeptide consisting of SEQ ID NOS: 3, 7, 13, or 20. An antibody specific for the above SEQ ID NOS: binds full-length proteins, truncated proteins, or protein fragments comprising the respective SEQ ID. The invention further provides an antibody that specifically binds canine apolipoprotein C-I, apolipoprotein C-II, fibrinogen alpha chain, or fibrinogen A-alpha chain. The invention further provides an antibody that specifically binds canine CysB1, Cys A, Kininogen, Inter-Alpha Inhibitor H4 (ITIH4), or keratin type I cytoskeletal 10. The antibody can be a monoclonal antibody, polyclonal antibody, antigen-binding antibody fragment, or a single chain antibody. 
     Another embodiment of the invention provides a method of diagnosing renal disease in a subject. The method comprises obtaining a biological sample from the subject; contacting the biological sample with an antibody specific for one or a plurality of SEQ ID NOS: 1-59 under conditions that allow polypeptide/antibody complexes to form; and detecting the levels of polypeptide/antibody complexes relative to levels present in control samples. In a preferred embodiment, a diagnostic antibody is specific for one or a plurality of SEQ ID NOS: 3, 7, 13, or 20, wherein the antibodies respectively specifically bind apolipoprotein C-I, apolipoprotein C-II, fibrinogen alpha chain, or fibrinogen A-alpha chain. The invention further provides an antibody that specifically binds canine Cystatin B, Cystatin A, Kininogen, Inter-Alpha Inhibitor H4 (ITIH4), or keratin type I cytoskeletal 10. 
     Yet another embodiment of the invention provides a method of detecting renal failure by identifying one or a plurality of polypeptides specific to SEQ ID NOS: 1-59 in a sample. The method comprises contacting antibodies that specifically bind to a polypeptide consisting of SEQ ID NOS: 1-59 with the sample under conditions that allow polypeptide/antibody complexes to form; and detecting the polypeptide/antibody complexes, wherein the differential levels of polypeptide/antibody complexes formed with patient sample versus control sample is an indication of renal disease. In an alternative embodiment, the method comprises contacting antibodies that specifically bind SEQ ID NOS: 3, 7, 13, or 20, wherein the antibodies respectively specifically bind apolipoprotein C-I, apolipoprotein C-II, fibrinogen alpha chain, or fibrinogen A-alpha chain. In yet another embodiment the antibodies specifically bind full-length proteins, truncated proteins, or protein fragments containing the respective SEQ ID. 
     The detection of the levels of polypeptide/antibody complexes present in the sample at differential levels to those of control samples (i.e., non-diseased) is an indication renal disease. In one embodiment of the invention the levels of polypeptide/antibody complexes in a patient sample at greater levels than controls is an indication of disease. In an alternative embodiment, the levels of polypeptide/antibody complexes in a patient at levels less than control is an indication of disease, particularly for inosine-specific antibodies. The antibodies can be monoclonal antibodies, polyclonal antibodies, antigen-binding antibody fragments, or single chain antibodies. The antibodies can specifically full-length proteins, truncated proteins, or protein fragments containing the respective SEQ ID NOS. In certain embodiments the inventive methods use metabolomics (i.e., LC/MS), and the biomarkers identified thereby, provide a significant improvement over current methods of detection. Instead of analyzing a solid tissue sample, cellular products or proteins are identified in patient biofluid or serum samples. This type of testing could reduce patient discomfort, permit repeated measurement, and allow more timely assessments. 
     One embodiment of the invention provides for one or a plurality of purified polypeptide comprising SEQ ID NOS: 1-59, wherein the polypeptide consists of less than about 40, 30, 20, or 10 contiguous naturally occurring amino acids; SEQ ID NOS: 1-3, wherein the polypeptide consists of less than about 30 contiguous naturally occurring apolipoprotein C-I amino acids; SEQ ID NOS: 4-7, wherein the polypeptide consists of less than about 40 contiguous naturally occurring fibrinogen A-alpha chain amino acids; SEQ ID NOS: 8-13, wherein the polypeptide consists of less than about 40 contiguous naturally occurring apolipoprotein C-II amino acids; or SEQ ID NOS: 14-20, wherein the polypeptide consists of less than about 20 contiguous naturally occurring fibrinogen alpha chain amino acids; SEQ ID NOS: 21-24, wherein the polypeptide consists of less than about 20 contiguous naturally occurring Kininogen chain amino acids; SEQ ID NOS: 25-28, wherein the polypeptide consists of less than about 30 contiguous naturally occurring Inter-Alpha Inhibitor H4 (ITIH4) chain amino acids; SEQ ID NOS: 29-31, wherein the polypeptide consists of less than about 20 contiguous naturally occurring CysA chain amino acids; SEQ ID NOS: 32-38, wherein the polypeptide consists of less than about 20 contiguous naturally occurring CysB1 chain amino acids; SEQ ID NOS: 39-59, wherein the polypeptide consists of less than about 30 contiguous naturally occurring keratin Type I cytoskeletol 10 chain amino acids. The invention also provides isolated polynucleotides that encode the purified polypeptide of the invention. 
     Therefore, the invention provides compositions and methods for the detecting, diagnosing, or prognosing renal disease. 
     Specific embodiments of this invention will become evident from the following more detailed description of certain preferred embodiments and the claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       These and other objects and features of this invention will be better understood from the following detailed description taken in conjunction with the drawings wherein: 
         FIG.  1    is a graph representing LC/MS measurement of inosine levels between high creatinine and control (low creatinine) dogs. 
         FIG.  2    is a graph representing inosine, NGAL, and creatinine levels over time in an induced canine model of renal disease. Units of measurement include: Inosine in μg/deciliter; Creatinine in mg/centiliter; and NGAL in ng/ml. 
         FIG.  3    is a series of graphs representing relative concentrations of apolipoprotein C1 ( FIG.  3 A ), kininogen ( FIG.  3 B ), and Inter-Alpha Inhibitor H4 (ITIH4) ( FIG.  3 C ) levels over time in an induced model of canine model of renal disease. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     This invention is more particularly described below and the Examples set forth herein are intended as illustrative only, as numerous modifications and variations therein will be apparent to those skilled in the art. As used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference unless the context clearly dictates otherwise. The terms used in the specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Some terms have been more specifically defined below to provide additional guidance to the practitioner regarding the description of the invention. 
     In other embodiments, the invention provides methods for detecting the polypeptides provided in Table 1, wherein the relative levels of the disclosed polypeptides identifies patients with renal disease. In the application and practice of these inventive methods, any method known in the art for detecting polypeptides can be used. In certain embodiments, these methods are practiced by identifying expression levels of full-length protein and polypeptide fragments of apolipoprotein C-I, apolipoprotein C-II, fibrinogen alpha chain, or fibrinogen A-alpha chain, CysB1, Cys A, Kininogen, Inter-Alpha Inhibitor H4 (ITIH4), or keratin type I cytoskeletal 10 in patient samples, wherein differential expression of the proteins as compared to a control are an indication of renal disease. In alternative embodiments, immunohistochemical (IHC) methods are used to detect renal disease in kidney biopsies. 
     In a particular embodiment, the invention provides methods for detecting inosine levels and other protein/metabolite levels in patient samples relative to controls. Relative levels can be measured by LC/MS (liquid chromatography/mass spectrometry). Alternatively, inosine and/or protein levels can be assessed with specific antibodies. For anti-inosine antibodies, see, Inouye, H. et al., Biochim Biophys Acta 1971, 240:594-603; Bonavida, B. et al., Immunochemistry 1972, 9:443-49; Inouye, H. et al., J Biol Chem 1973, 23:8125-29. Reduced levels of inosine are indicative of kidney/renal disease. 
     As used herein, a “patient” or “subject” to be treated by the disclosed methods can mean either a human or non-human animal but in certain particular embodiments is a human feline, or canine. 
     The term “patient sample” as used herein includes but is not limited to a blood, serum, plasma, or urine sample obtained from a patient. 
     The term “control sample” as used herein can mean a sample obtained from a non-diseased individual or population, more particularly an individual or population that does not suffer from renal disease. 
     The term “polypeptides” can refer to one or more of one type of polypeptide (a set of polypeptides). “Polypeptides” can also refer to mixtures of two or more different types of polypeptides (i.e., a mixture of polypeptides that includes but is not limited to full-length protein, truncated protein, or protein fragments). The terms “polypeptides” or “polypeptide” can each also mean “one or more polypeptides.” 
     The term “full-length” as used herein refers to a protein comprising its natural amino acid sequence as expressed in vivo, or variants thereof. The term “truncated” refers to a protein that is lacks amino acids from the N- or C-terminal ends of the protein. The term “peptide fragment” refers to a partial amino acid sequence from a larger protein. In particular embodiments, a peptide fragment is 10, 20, 30, 40, or 50 amino acids in length. 
     As disclosed herein, the polypeptides identified and provided by this invention comprise one or a plurality of proteins that have altered expression (e.g., either increased or decreased) in patients with renal disease. In certain embodiments, aberrant levels of the polypeptides set forth herein are associated with renal dysfunction; in particular, increased apolipoprotein C-I, increased apolipoprotein C-II, decreased fibrinogen A-alpha chain, or decreased fibrinogen alpha chain polypeptide fragments as detected inter alia by antibodies specific to the polypeptides of the invention. In certain embodiments aberrant levels of additional polypeptides and proteins are included and in particular inosine metabolite and the following proteins: apolipoprotein C-I, apolipoprotein C-II, fibrinogen alpha chain, or fibrinogen A-alpha chain, kininogen, and Inter-Alpha Inhibitor H4 (ITIH4). In some embodiments, the proteins are found in blood, serum, plasma, or urine. The relative levels of specific polypeptides can indicate progression of renal failure and disease severity. 
     In either embodiment, altered protein expression is relative to control (e.g., non-renal diseased) sample comprising the invention show differential expression levels as compared to control samples. This invention provides antibodies specific to the polypeptides of Table 1 and methods of use thereof for identifying renal disease, in patient samples and to provide prognosis and diagnosis thereby. It is an advantage of this invention that altered expression of the polypeptides provided herein can be readily detected using methods well known to the skilled worker. 
     In particular embodiments, the invention provides reagents and methods for identifying renal disease in a mammal, and more particularly, in dogs, cats and humans. In certain embodiments, the invention provides methods for providing a diagnosis and prognosis for a renal patient. As disclosed herein, identifying the polypeptides of this invention in patient samples can be an independent predictor of kidney disease or an identifier of disease stage (e.g., stages 1-5). This invention advantageously permits diagnosis and identification of kidney disease stage prior to stage three and is not limited by patient age or body mass. Accordingly, additional embodiments of the invention are directed to using said renal patient prognosis determined using the polypeptides of the invention to select appropriate renal therapies. 
     For the purposes of this invention, the term “immunological reagents” is intended to encompass antisera and antibodies, particularly monoclonal antibodies, as well as fragments thereof (including F(ab), F(ab) 2 , F(ab)′ and F v  fragments). Also included in the definition of immunological reagent are chimeric antibodies, humanized antibodies, and recombinantly-produced antibodies and fragments thereof. Immunological methods used in conjunction with the reagents of the invention include direct and indirect (for example, sandwich-type) labeling techniques, immunoaffinity columns, immunomagnetic beads, fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assays (ELISA), radioimmune assay (RIA), as well as peroxidase labeled secondary antibodies that detect the primary antibody. 
     The immunological reagents of the invention are preferably detectably-labeled, most preferably using fluorescent labels that have excitation and emission wavelengths adapted for detection using commercially-available instruments such as and most preferably fluorescence activated cell sorters. Examples of fluorescent labels useful in the practice of the invention include phycoerythrin (PE), fluorescein isothiocyanate (FITC), rhodamine (RH), Texas Red (TX), Cy3, Hoechst 33258, and 4′,6-diamidino-2-phenylindole (DAPI). Such labels can be conjugated to immunological reagents, such as antibodies and most preferably monoclonal antibodies using standard techniques (Maino et al., 1995,  Cytometry  20: 127-133). 
     Antibodies of the invention are antibody molecules that specifically bind to polypeptides of the invention as provided in Table 1, variant polypeptides of the invention, or fragments thereof. An antibody of the invention can be specific for polypeptide fragments of apolipoprotein C-I, apolipoprotein C-II, fibrinogen alpha chain, or fibrinogen A-alpha chain, for example, an antibody specific for one or a plurality of SEQ ID NOS: 3, 7, 13, or 20. An antibody of the invention preferably recognizes multiple protein products. For example an antibody specific to SEQ ID NO: 3 recognizes multiple peptide fragment of apolipoprotein C-I, including SEQ ID NOS: 1-2, as well as full-length protein. One of skill in the art can easily determine if an antibody is specific for a polypeptide of Table 1 using assays described herein. An antibody of the invention can be a polyclonal antibody, a monoclonal antibody, a single chain antibody (scFv), or an antigen binding fragment of an antibody. Antigen-binding fragments of antibodies are a portion of an intact antibody comprising the antigen binding site or variable region of an intact antibody, wherein the portion is free of the constant heavy chain domains of the Fc region of the intact antibody. Examples of antigen binding antibody fragments include Fab, Fab′, Fab′-SH, F(ab′) 2  and F v  fragments. 
     An antibody of the invention can be any antibody class, including for example, IgG, IgM, IgA, IgD and IgE. An antibody or fragment thereof binds to an epitope of a polypeptide of the invention. An antibody can be made in vivo in suitable laboratory animals or in vitro using recombinant DNA techniques. Means for preparing and characterizing antibodies are well know in the art. See, e.g., Dean,  Methods Mol. Biol.  80:23-37 (1998); Dean,  Methods Mol. Biol.  32:361-79 (1994); Baileg,  Methods Mol. Biol.  32:381-88 (1994); Gullick,  Methods Mol. Biol.  32:389-99 (1994); Drenckhahn et al.  Methods Cell. Biol.  37:7-56 (1993); Morrison,  Ann. Rev. Immunol.  10:239-65 (1992); Wright et al.  Crit. Rev. Immunol.  12:125-68 (1992). For example, polyclonal antibodies can be produced by administering a polypeptide of the invention to an animal, such as a human or other primate, mouse, rat, rabbit, guinea pig, goat, pig, dog, cow, sheep, donkey, or horse. Serum from the immunized animal is collected and the antibodies are purified from the plasma by, for example, precipitation with ammonium sulfate, followed by chromatography, such as affinity chromatography. Techniques for producing and processing polyclonal antibodies are known in the art. 
     “Specifically binds,” “specifically bind,” or “specific for” means that a first antigen, e.g., a polypeptide of Table 1, recognizes and binds to an antibody of the invention with greater affinity than to other, non-specific molecules. “Specifically binds,” “specifically bind” or “specific for” also means a first antibody, e.g., an antibody raised against SEQ ID NOS:1-59, recognizes and binds to SEQ ID NOS:1-59, with greater affinity than to other non-specific molecules. A non-specific molecule is an antigen that shares no common epitope with the first antigen. Specific binding can be tested using, for example, an enzyme-linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), or a western blot assay using methodology well known in the art. 
     The phrase “competes for binding” as used herein refers to an antibody that has a binding affinity for a particular polypeptide sequence or antigen such that when present, it will bind preferentially and specifically to the peptide sequence/antigen over other non-specific molecules. Again, a non-specific molecule is an antigen that shares no common epitope with the first antigen. 
     Antibodies of the invention include antibodies and antigen binding fragments thereof that (a) compete with a reference antibody for binding to SEQ ID NOS: 1-59 or antigen binding fragments thereof; (b) binds to the same epitope of SEQ ID NOS: 1-59 or antigen binding fragments thereof as a reference antibody; (c) binds to SEQ ID NOS: 1-59 or antigen binding fragments thereof with substantially the same K d  as a reference antibody; and/or (d) binds to SEQ ID NOS: 1-59 or fragments thereof with substantially the same off rate as a reference antibody, wherein the reference antibody is an antibody or antigen-binding fragment thereof that specifically binds to a polypeptide of SEQ ID NOS: 1-59 or antigen binding fragments thereof with a binding affinity K a  of 10 7  l/mol or more. 
     The affinity of a molecule X for its partner Y can be represented by a dissociation constant (Kd). The equilibrium dissocation constant (Kd) is calculated at the ration of k off /k on . See Chen, Y. et al., 1999, J. Mol. Biol. 293: 865-881. A variety of methods are known in the art for measuring affinity constants, which can be used for purposes of the present invention. In a particular embodiment, the reference antibody is an antibody or antigen-binding fragment thereof that has a binding affinity to a polypeptide of SEQ ID NOS: 1-59 with a particular K on  rate/association rate or K off  rate. In one embodiment, the antibodies of the invention specifically bind with a K on  of 6×10 5  M −1 s −1  or better; antibodies specifically bind with a K off  rate of 5×10 −6  s −1  or better; or antibodies specifically binds with a binding affinity of 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM or better. 
     Additionally, monoclonal antibodies directed against epitopes present on a polypeptide of the invention can also be readily produced. For example, normal B cells from a mammal, such as a mouse, which was immunized with a polypeptide of the invention can be fused with, for example, HAT-sensitive mouse myeloma cells to produce hybridomas. Hybridomas producing polypeptide-specific antibodies can be identified using RIA or ELISA and isolated by cloning in semi-solid agar or by limiting dilution. Clones producing specific antibodies are isolated by another round of screening. Monoclonal antibodies can be screened for specificity using standard techniques, for example, by binding a polypeptide of the invention to a microtiter plate and measuring binding of the monoclonal antibody by an ELISA assay. Techniques for producing and processing monoclonal antibodies are known in the art. See e.g., Kohler &amp; Milstein, Nature, 256:495 (1975). Particular isotypes of a monoclonal antibody can be prepared directly, by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of a different isotype by using a sib selection technique to isolate class-switch variants. See Steplewski et al.,  P.N.A.S. U.S.A.  82:8653 1985; Spria et al.,  J. Immunolog. Meth.  74:307, 1984. Monoclonal antibodies of the invention can also be recombinant monoclonal antibodies. See, e.g., U.S. Pat. Nos. 4,474,893; 4,816,567. Antibodies of the invention can also be chemically constructed. See, e.g., U.S. Pat. No. 4,676,980. 
     Antibodies of the invention can be chimeric (see, e.g., U.S. Pat. No. 5,482,856), humanized (see, e.g., Jones et al.,  Nature  321:522 (1986); Reichmann et al.,  Nature  332:323 (1988); Presta,  Curr. Op. Struct. Biol.  2:593 (1992)), caninized, canine, or human antibodies. Human antibodies can be made by, for example, direct immortilization, phage display, transgenic mice, or a Trimera methodology, see e.g., Reisener et al.,  Trends Biotechnol.  16:242-246 (1998). 
     Antibodies that specifically bind SEQ ID NOS: 1-59 are particularly useful for detecting the presence of polypeptide fragments specific for renal disease present in a sample, such as a serum, blood, plasma, cell, tissue, or urine sample from an animal. An immunoassay for can utilize one antibody or several antibodies. An immunoassay can use, for example, a monoclonal antibody specific for one epitope, a combination of monoclonal antibodies specific for epitopes of one polypeptide, monoclonal antibodies specific for epitopes of different polypeptides, polyclonal antibodies specific for the same antigen, polyclonal antibodies specific for different antigens, or a combination of monoclonal and polyclonal antibodies. Immunoassay protocols can be based upon, for example, competition, direct reaction, or sandwich type assays using, for example, labeled antibody. Antibodies of the invention can be labeled with any type of label known in the art, including, for example, fluorescent, chemiluminescent, radioactive, enzyme, colloidal metal, radioisotope and bioluminescent labels. 
     Antibodies of the invention or antigen-binding fragments thereof can be bound to a support and used to detect the presence of proteins differential produced in renal disease. Supports include, for example, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magletite. 
     Antibodies of the invention can further be used to isolate polypeptides by immunoaffinity columns. The antibodies can be affixed to a solid support by, for example, absorption or by covalent linkage so that the antibodies retain their immunoselective activity. Optionally, spacer groups can be included so that the antigen binding site of the antibody remains accessible. The immobilized antibodies can then be used to bind the polypeptides of Table 1 from a biological sample, including but not limited to saliva, serum, blood, and urine. 
     Antibodies of the invention can also be used in immunolocalization studies to analyze the presence and distribution of a polypeptide of the invention during various cellular events or physiological conditions. Antibodies can also be used to identify molecules involved in passive immunization and to identify molecules involved in the biosynthesis of non-protein antigens. Identification of such molecules can be useful in vaccine development. Antibodies of the invention, including, for example, monoclonal antibodies and single chain antibodies, can be used to monitor the course of amelioration of a kidney disease. By measuring the increase or decrease of antibodies specific for the polypeptides of Table 1 in a test sample from an animal, it can be determined whether a particular therapeutic regiment aimed at ameliorating the disorder is effective. Antibodies can be detected and/or quantified using for example, direct binding assays such as RIA, ELISA, or Western blot assays. 
     The methods of the invention can be used to detect polypeptide fragments of Table 1 or full-length proteins containing an amino acid sequence provided in Table 1, wherein antibodies or antigen-binding antibody fragments are specific for SEQ ID NOS: 1-59. A biological sample can include, for example, sera, blood, cells, plasma, saliva, or urine from a mammal such as a dog, cat or human. The test sample can be untreated, precipitated, fractionated, separated, diluted, concentrated, or purified. 
     In one embodiment methods of the invention comprise contacting a test sample with one or a plurality of antibodies specific to SEQ ID NOS: 1-59 under conditions that allow polypeptide/antibody complexes, i.e., immunocomplexes, to form. That is, antibodies of the invention specifically bind to one or a plurality of polypetides of SEQ ID NOS: 1-59 located in the sample. One of skill in the art is familiar with assays and conditions that are used to detect antibody/polypeptide complex binding. The formation of a complex between polypeptides and antibodies in the sample is detected. The formation of antibody/polypeptide complexes is an indication that polypeptides are present in the patient sample. 
     Antibodies of the invention can be used in a method of the diagnosis renal disease by obtaining a test sample from, e.g., a human, cat or dog suspected of suffering from renal disease. The test sample is contacted with antibodies of the invention under conditions enabling the formation of antibody-antigen complexes (i.e., immunocomplexes). One of skill in the art is aware of conditions that enable and are appropriate for formation of antigen/antibody complexes. The amount of antibody-antigen complexes can be determined by methodology known in the art. 
     Methods of the invention comprise diagnosing renal disease in a patient by identifying the differential expression of the polypeptides of Table 1 in a patient sample as compared to control. These methods include the diagnosis or identification of disease stage (e.g., stages 1-5). The present invention further include methods for prognosing patient health, monitoring disease progression, and/or assessing/monitoring treatment efficacy by identifying levels of specific polypeptides of the invention in a patient sample. In one aspect, the inventive methods can be performed at multiple time points to evaluate disease progression or treatment efficacy. In a particular embodiment, the methods may be performed at diagnosis and then at specific time points post-treatment wherein a specific therapy should result in a reduction or amelioration of disease progression. 
     In an alternative embodiment, the methods of the invention are used to assess the efficacy of a composition or treatment regime (whether a composition or diet) for the amelioration of renal disease progression. Similarly, the methods of the invention can be used for assessing a composition or treatment regimens activity on patient levels of the polypeptides of Table 1. 
     Differential levels of antibody-complexes present in patient samples versus control samples provides an indicator for renal disease. In one embodiment of the invention an antibody is specific for one or plurality of the polypeptides provided in Table 1. An antibody of the invention can be contacted with a test sample. Antibodies specific to the polypeptides present in a test sample will form antigen-antibody complexes under suitable conditions. The amount of antibody-antigen complexes can be determined by methods known in the art. 
     In one embodiment of the invention, renal disease can be detected in a subject. A biological sample is obtained from the subject. One or more antibodies specific to the polypeptides comprising SEQ ID NOS:1-59 or other polypeptides of the invention are contacted with the biological sample under conditions that allow polypeptide/antibody complexes to form. The polypeptide/antibody complexes are detected. The detection of the polypeptide/antibody complexes at differential levels as compared to controls is an indication that the mammal has renal disease. 
     In one embodiment of the invention, the polypeptide/antibody complex is detected when an indicator reagent, such as an enzyme conjugate, which is bound to the antibody, catalyzes a detectable reaction. Optionally, an indicator reagent comprising a signal generating compound can be applied to the polypeptide/antibody complex under conditions that allow formation of a polypeptide/antibody/indicator complex. The polypeptide/antibody/indicator complex is detected. Optionally, the polypeptide or antibody can be labeled with an indicator reagent prior to the formation of a polypeptide/antibody complex. The method can optionally comprise a positive or negative control. 
     In one embodiment of the invention, one or more antibodies of the invention are attached to a solid phase or substrate. A test sample potentially comprising a protein comprising a polypeptide of the invention is added to the substrate. One or more antibodies that specifically bind polypeptides of the invention are added. The antibodies can be the same antibodies used on the solid phase or can be from a different source or species and can be linked to an indicator reagent, such as an enzyme conjugate. Wash steps can be performed prior to each addition. A chromophore or enzyme substrate is added and color is allowed to develop. The color reaction is stopped and the color can be quantified using, for example, a spectrophotometer. 
     In another embodiment of the invention, one or more antibodies of the invention are attached to a solid phase or substrate. A test sample potentially containing a protein comprising a polypeptide of the invention is added to the substrate. Second anti-species antibodies that specifically bind polypeptides of the invention are added. These second antibodies are from a different species than the solid phase antibodies. Third anti-species antibodies are added that specifically bind the second antibodies and that do not specifically bind the solid phase antibodies are added. The third antibodies can comprise and indicator reagent such as an enzyme conjugate. Wash steps can be performed prior to each addition. A chromophore or enzyme substrate is added and color is allowed to develop. The color reaction is stopped and the color can be quantified using, for example, a spectrophotometer. 
     In one embodiment, one or more capture antibodies can specifically bind to one or more epitopes of a polypeptide of the invention. The capture antibody or antibodies would be used to immobilize one or a plurality of polypeptide of SEQ ID NOS: 1-59 to, for example a solid support. One or more detection antibodies can specifically bind to the same one or more epitopes or different one or more epitopes of the polypeptides of the invention. The detection antibody can be used to detect or visualize the immobilization of the polypeptide of the invention to a solid support. This embodiment is advantageous because it is more specific and more sensitive than assays using only one antibody for both capture and detection functions. 
     Assays of the invention include, but are not limited to those based on competition, direct reaction or sandwich-type assays, including, but not limited to enzyme linked immunosorbent assay (ELISA), western blot, IFA, radioimmunoassay (RIA), hemagglutination (HA), fluorescence polarization immunoassay (FPIA), and microtiter plate assays (any assay done in one or more wells of a microtiter plate). One assay of the invention comprises a reversible flow chromatographic binding assay, for example a SNAP® assay. See e.g., U.S. Pat. No. 5,726,010. 
     Assays can use solid phases or substrates or can be performed by immunoprecipitation or any other methods that do not utilize solid phases. Where a solid phase or substrate is used, one or more polypeptides of the invention are directly or indirectly attached to a solid support or a substrate such as a microtiter well, magnetic bead, non-magnetic bead, column, matrix, membrane, fibrous mat composed of synthetic or natural fibers (e.g., glass or cellulose-based materials or thermoplastic polymers, such as, polyethylene, polypropylene, or polyester), sintered structure composed of particulate materials (e.g., glass or various thermoplastic polymers), or cast membrane film composed of nitrocellulose, nylon, polysulfone or the like (generally synthetic in nature). In one embodiment of the invention a substrate is sintered, fine particles of polyethylene, commonly known as porous polyethylene, for example, 10-15 micron porous polyethylene from Chromex Corporation (Albuquerque, N. Mex.). All of these substrate materials can be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics. Suitable methods for immobilizing antibodies on solid phases include ionic, hydrophobic, covalent interactions and the like. 
     In one type of assay format, one or more antibodies can be coated on a solid phase or substrate. A test sample suspected of containing polypeptides of Table 1 or fragments thereof is incubated with an indicator reagent comprising a signal generating compound conjugated to an antibodies or antibody fragments specific for said polypeptides for a time and under conditions sufficient to form antigen/antibody complexes of either antibodies of the solid phase to the test sample polypeptides or the indicator reagent compound conjugated to an antibody specific for the polypeptides. The binding of the indicator reagent conjugated to anti-polypeptide antibodies to the solid phase can be quantitatively measured. A measurable alteration in the signal compared to the signal generated from a control sample indicates the presence of polypeptides of the present invention (SEQ ID NOS: 1-59). This type of assay can quantitate the amount of polypeptide in a test sample. 
     In another type of assay format, one or more antibodies of the invention are coated onto a support or substrate. An antibody of the invention is conjugated to an indicator reagent and added to a test sample. This mixture is applied to the support or substrate. If polypeptides of the invention are present in the test sample, they will bind the one or more antibodies conjugated to an indicator reagent and to the one or more antibodies immobilized on the support. The polypeptide/antibody/indicator complex can then be detected. This type of assay can quantitate the amount of polypeptide in a test sample. 
     In another type of assay format, one or more antibodies of the invention are coated onto a support or substrate. The test sample is applied to the support or substrate and incubated. Unbound components from the sample are washed away by washing the solid support with a wash solution. If the polypeptides of Table 1 are present in the test sample, they will bind to the antibody coated on the solid phase. This polypeptide/antibody complex can be detected using a second species-specific antibody that is conjugated to an indicator reagent. The polypeptide/antibody/anti-species antibody indicator complex can then be detected. This type of assay can quantitate the amount of polypeptides in a test sample. 
     The formation of a polypeptide/antibody complex or a polypeptide/antibody/indicator complex can be detected by, for example, radiometric, colorimetric, fluorometric, size-separation, or precipitation methods. Optionally, detection of a polypeptide/antibody complex is by the addition of a secondary antibody that is coupled to an indicator reagent comprising a signal generating compound. Indicator reagents comprising signal generating compounds (labels) associated with a polypeptide/antibody complex can be detected using the methods described above and include chromogenic agents, catalysts such as enzyme conjugates fluorescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums, ruthenium, and luminol, radioactive elements, direct visual labels, as well as cofactors, inhibitors, magnetic particles, and the like. Examples of enzyme conjugates include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances. 
     Formation of the complex at differential levels as compared to control is indicative of the presence of renal disease. Therefore, the methods of the invention can be used to diagnose kidney disease in an animal. 
     The phrase “determining the amounts” as used herein refers to measuring or identifying the levels of one or a plurality polypeptides in a patient sample. In a particular embodiment, the identification of a specific epitope in polypeptides of multiple lengths including full-length protein, truncated protein, and protein fragments is provided. This can be accomplished by methodology well known in the art for the detection of polypeptides including using antibodies including, for example enzyme-linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), or a western blot assay, or immunohistochemistry. Alternatively polypeptides of the present invention, SEQ ID NOS: 1-59, can be determined by mass spectrometry or similar methods known by one of skill in the art. Determining the amount of polypeptide present in a patient sample is accomplished by such in vitro analysis and experimental manipulation. The amount of polypeptide present cannot be assessed by mere inspection. 
     In an alternative embodiment, elevated or reduced levels of one or a plurality of polypeptide transcripts of Table 1 present in a patient sample are detected by a process of hybridizing a nucleic acid probe that selectively hybridizes to the polypeptides of the invention. Conditions are utilized that permit high stringency hybridization between the nucleic acid probe, which is used as a detection means, and the polypeptide transcripts of the invention, wherein a level of nucleic acid complex formation and detection is indicative of the level of transcript in a sample. The enhanced or reduced level of polypeptide is indicative of renal disease. Methods for producing nucleic acid probes specific to the polypeptide transcripts are well known in the art. 
     The methods of the invention can also indicate the amount or quantity of polypeptides of Table 1 or full-length proteins comprising a polypeptide sequence of Table 1 in a test sample. In a particular embodiment, the amount or quantity of certain polypeptides provides an indicator of disease stage (i.e., stages 1-5), disease progression, and/or a prognostic indicator. With many indicator reagents, such as enzyme conjugates, the amount of polypeptide present is proportional to the signal generated. Depending upon the type of test sample, it can be diluted with a suitable buffer reagent, concentrated, or contacted with a solid phase without any manipulation. For example, it usually is preferred to test serum or plasma samples that previously have been diluted, or concentrated specimens such as urine, in order to determine the presence and/or amount of polypeptide present. 
     Polypeptides and assays of the invention can be combined with other polypeptides or assays to detect the presence of renal disease. For example, polypeptides and assays of the invention can be combined with reagents that creatinine or general protein levels. 
     The invention also provides kits for performing the methods disclosed herein. In certain embodiments, the kits of this invention comprise one or a plurality of antibodies specific for one or plurality of the polypeptides provided in Table 1, wherein in particular embodiments said antibody are monoclonal antibodies, polyclonal antibodies, antigen-binding antibody fragments, or single chain antibodies. Optionally included in specific embodiments of the kits of the invention can be instructions for use, as well as secondary antibodies useful inter alia in sandwich assays understood by those in the art. Distinguishingly labeled embodiments of the antibody components of said kits, as well as reagents and methods for labeling said antibodies, are also advantageously-provided components of the kits of the invention. 
     In further embodiments, kits of the invention comprise one or plurality of antibodies that each specifically bind to differential protein expression of one or a plurality of the polypeptides identified in Table 1. In certain embodiments, said antibodies are provided on a solid support, including without limitation chips, microarrays, beads and the like. Optionally included in specific embodiments of the kits of the invention can be instructions for use, as well as secondary antibodies useful inter alia in sandwich assays understood by those in the art. Distinguishingly labeled embodiments of the antibody components of said kits, as well as reagents and methods for labeling said antibodies, are also advantageously-provided components of the kits of the invention. 
     The kits of the present invention (e.g., articles of manufacture) are for detecting the polypeptides of Table 1, or protein fragment thereof in a patient sample. A kit comprises one or more antibodies of the invention and means for determining binding of the antibodies to full-length proteins or protein fragments containing the amino acid sequences provided in Table 1 present in the sample. A kit or article of manufacture can also comprise one or more antibodies or antibody fragments of the invention and means for determining binding of the antibodies or antibody fragments to polypeptides in the sample. A kit can comprise a device containing one or more polypeptides or antibodies of the invention and instructions for use of the one or more polypeptides or antibodies for, e.g., the identification of renal disease in a mammal. The kit can also comprise packaging material comprising a label that indicates that the one or more polypeptides or antibodies of the kit can be used for the identification of kidney dysfunction. Other components such as buffers, controls, and the like, known to those of ordinary skill in art, can be included in such test kits. The polypeptides, antibodies, assays, and kits of the invention are useful, for example, in the diagnosis of individual cases of renal disease in a patient. 
     The kits of the invention are useful for diagnosing, prognosing, or monitoring the treatment of renal disease, particularly canine renal disease. 
     One embodiment provides a purified polypeptide comprising SEQ ID NOS:1-59, wherein the polypeptide consists of less than about 50, 40, 35, 30, 25, 20, 15, 10 (or any range between about 31 and about 175) contiguous naturally occurring amino acids. In one embodiment of the invention a purified polypeptide consists of more than about 10, 15, 20, 25, 30, 35, 40, 50, 60, contiguous naturally occurring amino acids. 
     The fact that polypeptides SEQ ID NOS:1-59 are smaller than the full length proteins is important because smaller polypeptides can have greater specificity and/or sensitivity than full length polypeptides assays. These smaller polypeptides can be less expensive to manufacture, and may be obtained at greater purity than the full length polypeptide. Additionally, the smaller fragments and the levels of smaller fragments present in a sample are indicative of disease state. The differential expression of fragmented proteins is a marker for renal disease. 
     Variant polypeptides are at least about 80%, or about 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the polypeptide sequences shown in SEQ ID NOS:1-59 and are also polypeptides of the invention. For example, a variant polypeptide of SEQ ID NOS:1-59 can be about at least 97%, 94%, 90%, 87%, 84%, or 81% identical to SEQ ID NOS:1-59. Variant polypeptides have one or more conservative amino acid variations or other minor modifications and retain biological activity, i.e., are biologically functional equivalents. A biologically active equivalent has substantially equivalent function when compared to the corresponding wild-type polypeptide. In one embodiment of the invention a polypeptide has about 1, 2, 3, 4, 5, 10, 20 or less conservative amino acid substitutions. 
     Percent sequence identity has an art recognized meaning and there are a number of methods to measure identity between two polypeptide or polynucleotide sequences. See, e.g., Lesk, Ed.,  Computational Molecular Biology,  Oxford University Press, New York, (1988); Smith, Ed.,  Biocomputing: Informatics And Genome Projects,  Academic Press, New York, (1993); Griffin &amp; Griffin, Eds.,  Computer Analysis Of Sequence Data, Part I,  Humana Press, New Jersey, (1994); von Heinje,  Sequence Analysis In Molecular Biology,  Academic Press, (1987); and Gribskov &amp; Devereux, Eds.,  Sequence Analysis Primer,  M Stockton Press, New York, (1991). Methods for aligning polynucleotides or polypeptides are codified in computer programs, including the GCG program package (Devereux et al.,  Nuc. Acids Res.  12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al.,  J. Molec. Biol.  215:403 (1990)), and Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) which uses the local homology algorithm of Smith and Waterman ( Adv. App. Math.,  2:482-489 (1981)). For example, the computer program ALIGN which employs the FASTA algorithm can be used, with an affine gap search with a gap open penalty of −12 and a gap extension penalty of −2. 
     When using any of the sequence alignment programs to determine whether a particular sequence is, for instance, about 95% identical to a reference sequence, the parameters are set such that the percentage of identity is calculated over the full length of the reference polynucleotide and that gaps in identity of up to 5% of the total number of nucleotides in the reference polynucleotide are allowed. 
     Variant polypeptides can generally be identified by modifying one of the polypeptide sequences of the invention, and evaluating the properties of the modified polypeptide to determine if it is a biological equivalent. A variant is a biological equivalent if it reacts substantially the same as a polypeptide of the invention in an assay such as an immunohistochemical assay, an enzyme-linked immunosorbent Assay (ELISA), a radioimmunoassay (RIA), immunoenzyme assay or a western blot assay, e.g. has 90-110% of the activity of the original polypeptide. In one embodiment, the assay is a competition assay wherein the biologically equivalent polypeptide is capable of reducing binding of the polypeptide of the invention to a corresponding reactive antigen or antibody by about 80, 95, 99, or 100%. An antibody that specifically binds a corresponding wild-type polypeptide also specifically binds the variant polypeptide. 
     A conservative substitution is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. 
     A polypeptide of the invention can further comprise a signal (or leader) sequence that co-translationally or post-translationally directs transfer of the protein. The polypeptide can also comprise a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide can be conjugated to an immunoglobulin Fc region or bovine serum albumin. 
     A polypeptide can be covalently or non-covalently linked to an amino acid sequence to which the polypeptide is not normally associated with in nature, i.e., a heterologous amino acid sequence. A heterologous amino acid sequence can be from a different organism, a synthetic sequence, or a sequence not usually located at the carboxy or amino terminus of a polypeptide of the invention. Additionally, a polypeptide can be covalently or non-covalently linked to compounds or molecules other than amino acids, such as indicator reagents. A polypeptide can be covalently or non-covalently linked to an amino acid spacer, an amino acid linker, a signal sequence, a stop transfer sequence, a transmembrane domain, a protein purification ligand, or a combination thereof. A polypeptide can also be linked to a moiety (i.e., a functional group that can be a polypeptide or other compound) that enhances an immune response (e.g., cytokines such as IL-2), a moiety that facilitates purification (e.g., affinity tags such as a six-histidine tag, trpE, glutathione, maltose binding protein), or a moiety that facilitates polypeptide stability (e.g., polyethylene glycol; amino terminus protecting groups such as acetyl, propyl, succinyl, benzyl, benzyloxycarbonyl or t-butyloxycarbonyl; carboxyl terminus protecting groups such as amide, methylamide, and ethylamide). In one embodiment of the invention a protein purification ligand can be one or more C amino acid residues at, for example, the amino terminus or carboxy terminus or both termini of a polypeptide of the invention. An amino acid spacer is a sequence of amino acids that are not associated with a polypeptide of the invention in nature. An amino acid spacer can comprise about 1, 5, 10, 20, 100, or 1,000 amino acids. 
     If desired, a polypeptide of the invention can be part of a fusion protein, which contains other amino acid sequences, such as amino acid linkers, amino acid spacers, signal sequences, TMR stop transfer sequences, transmembrane domains, as well as ligands useful in protein purification, such as glutathione-S-transferase, histidine tag, and  Staphylococcal  protein A. More than one polypeptide of the invention can be present in a fusion protein of the invention. A polypeptide of the invention can be operably linked to proteins of a different organism or to form fusion proteins. A fusion protein of the invention can comprise one or more of polypeptides of the invention, fragments thereof, or combinations thereof. A fusion protein does not occur in nature. The term “operably linked” means that the polypeptide of the invention and the other polypeptides are fused in-frame to each other either to the N-terminus or C-terminus of the polypeptide of the invention. 
     Polypeptides of the invention can be in a multimeric form. That is, a polypeptide can comprise one or more copies of a polypeptide of the invention or a combination thereof. A multimeric polypeptide can be a multiple antigen peptide (MAP). See e.g., Tam, J. Immunol. Methods, 196:17-32 (1996). 
     Polypeptides of the invention can comprise an antigen that is recognized by an antibody specific for the polypeptides of SEQ ID NOS: 1-59. The antigen can comprise one or more epitopes (i.e., antigenic determinants). An epitope can be a linear epitope, sequential epitope or a conformational epitope. Epitopes within a polypeptide of the invention can be identified by several methods. See, e.g., U.S. Pat. No. 4,554,101; Jameson &amp; Wolf,  CABIOS  4:181-186 (1988). For example, a polypeptide of the invention can be isolated and screened. A series of short peptides, which together span an entire polypeptide sequence, can be prepared by proteolytic cleavage. By starting with, for example, 30-mer polypeptide fragments (or smaller fragments), each fragment can be tested for the presence of epitopes recognized in an ELISA. For example, in an ELISA assay, a polypeptide of the invention, such as a 30-mer polypeptide fragment, is attached to a solid support, such as the wells of a plastic multi-well plate. A population of antibodies are labeled, added to the solid support and allowed to bind to the unlabeled antigen, under conditions where non-specific absorption is blocked, and any unbound antibody and other proteins are washed away. Antibody binding is detected by, for example, a reaction that converts a colorless substrate into a colored reaction product. Progressively smaller and overlapping fragments can then be tested from an identified 30-mer to map the epitope of interest. 
     A polypeptide of the invention can be produced recombinantly. A polynucleotide encoding a polypeptide of the invention can be introduced into a recombinant expression vector, which can be expressed in a suitable expression host cell system using techniques well known in the art. A variety of bacterial, yeast, plant, mammalian, and insect expression systems are available in the art and any such expression system can be used. Optionally, a polynucleotide encoding a polypeptide can be translated in a cell-free translation system. A polypeptide can also be chemically synthesized or obtained from patient samples or cells. 
     An immunogenic polypeptide of the invention can comprise an amino acid sequence shown in SEQ ID NOS:1-59 or fragments thereof. An immunogenic polypeptide can elicit antibodies or other immune responses (e.g., T-cell responses of the immune system) that recognize epitopes of a polypeptide having SEQ ID NOS:1-59. An immunogenic polypeptide of the invention can also be a fragment of a polypeptide that has an amino acid sequence shown in SEQ ID NOS:1-6. An immunogenic polypeptide fragment of the invention can be about 10, 15, 20, 25, 30, 40, 50 or more amino acids in length. An immunogenic polypeptide fragment of the invention can be about 50, 40, 30, 20, 15, 10 or less amino acids in length. 
     The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms, while retaining their ordinary meanings. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims. 
     Embodiments of the methods of this invention comprising the above-mentioned features are intended to fall within the scope of this invention. 
     EXAMPLES 
     The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They set forth for explanatory purposes only, and are not to be taken as limiting the invention. 
     Example 1 
     Identification and Purification of Blood Samples 
     Patient blood samples were collected from dogs. The dogs were members of a single family maintained at Texas A &amp; M University since 1997. More specifically, this family is a colony of heterozygous (carrier) females with X-linked hereditary nephropathy (XLHN). XLHN is caused by a mutation in the gene COL4A5 which in the female dogs causes a mosaic expression of type IV collagen peptides and onset of glomerular proteinuria between 3 and 6 months of age. Nabity et al., J Vet Intern Med 2007; 21:425-430. Control versus experimental (diseased) was selected wherein controls were healthy dogs and the experimental or diseased group were dogs exhibiting elevated creatinine levels. 
     The following procedure was utilized for the preparation of patient samples for experimental analysis. Utilizing a 0.5 mL protein LoBind eppendorf tube, 110 uL of serum was precipitated by addition of 200 uL N,N-dimethylacetamide, which was followed by vortexing for 10 seconds, and incubating the sample at room temperature for 30 minutes. Resulting precipitate was pelleted by centrifugation at 13000 rpm for 30 minutes at 10° C. Supernatant was decanted into a borosilicate culture tube containing 5.0 mL of 0.1% formic acid in water and mixed to homogeneity. 
     The diluted extract was then further fractionated using a Caliper Life Science Rapid trace automated solid phase extraction apparatus as follows: 1 mL (30 mg) Waters OASIS® HLB solid phase extraction cartridges were conditioned at 0.5 mL/sec first with 1.0 mL 0.1% formic acid in water followed by 1.0 mL 0.1% formic acid in acetonitrile and finally with 2.0 mL 0.1% formic acid in water. Samples were loaded at a flow rate of 0.015 mL/sec then washed with 1.25 mL of 0.1% formic acid in water at a flow rate of 0.015 mL/sec. 1.25 mL fractions were then collected into borosilicate glass tubes containing 5 μL of 20 mg/mL N-nonyl-β-glucopyranoside in water. Fractions were eluted consecutively and collected separately using first 0.1% formic acid in 35% acetonitrile/water and next 0.1% formic acid in 65% acetonitrile/water at a flow rate of 0.015 mL/sec. The canula and solvent transfer lines were purged and cleaned between runs with 3.0 mL of 0.1% formic acid in acetonitrile then 3.0 mL of 0.1% formic acid in water at a flow rate of 0.5 mL/sec. 
     Fractions were split in half and evaporated to dried state at room temperature using a Savant Speed Vac Concentrator Model SVC-100H. The dried samples were then separated into two batches and stored at −80° C. For analysis, the samples were reconstituted in 60 μL 0.1% formic acid in either 5% (35% fraction) or 35% (65% fraction) acetonitrile and analyzed by liquid chromatography/mass spectrometry (LC/MS). 
     Example 2 
     Identification of Polypeptides in Diseased Does by Liquid Chromatography/Mass Spectrometry 
     Experimental and control samples were subjected to liquid chromatography/mass spectrometry (LC/MS) for the identification of differentially produced polypeptides by mass. The identified polypeptide masses were then annotated to determine the corresponding protein name by performing a peptide ID search of existing databases. A unique databases for peptide annotation was created from NCBI, Swissprot, Uniprot. 
     The resulting data provided the polypeptides provided in Table 1. SEQ ID NOS: 1-59 are the polypeptides that were differentially produced in dogs with renal disease. Therefore, these polypeptides provide unique biomarkers for the detection renal disease in dogs. 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Polypeptides differentially produced in dogs with renal disease. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 Expression 
               
               
                 Accession 
                 No. Peptides 
                 No. AAs 
                 MW [kDa] 
                 Description 
                   
                 Levels 
               
               
                   
               
               
                 P56595 
                  3 
                   88 
                    9.7 
                 Apolipoprotein C-I 
                   
                 Increased 
               
               
                   
                   
                   
                   
                 OS =  Canis   
                   
                   
               
               
                   
                   
                   
                   
                 
                   familiaris 
                 
                   
                   
               
               
                   
                   
                   
                   
                 GN = APOC1 PE = 2 
                   
                   
               
               
                   
                   
                   
                   
                 SV = 1- 
                   
                   
               
               
                   
                   
                   
                   
                 [APOC1_CANFA] 
               
               
                   
               
               
                   
                 Sequence 
                 m/z [Da] 
                 MH+ [Da] 
                 RT [min] 
               
               
                   
               
               
                   
                 AGEISSTFERIPDKL 
                  965.83238 
                 2895.48259 
                   27.98 
                   
                   
               
               
                   
                 KEFGNTLEDKA 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 1) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 EISSTFERIPDKLKE 
                  923.14789 
                 2767.42910 
                   27.41 
                   
                   
               
               
                   
                 FGNTLEDKA 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 2) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 DKLKEFGNTLEDKA 
                  536.61725 
                 1607.83719 
                   20.36 
                   
                   
               
               
                   
                 (SEQ. ID NO: 3) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Q28243 
                  4 
                  443 
                   45.9 
                 Fibrinogen A- 
                   
                 Decreased 
               
               
                   
                   
                   
                   
                 alpha-chain 
                   
                   
               
               
                   
                   
                   
                   
                 (Fragment) 
                   
                   
               
               
                   
                   
                   
                   
                 OS =  Canis   
                   
                   
               
               
                   
                   
                   
                   
                   familiaris  PE = 4 
                   
                   
               
               
                   
                   
                   
                   
                 SV = 1- 
                   
                   
               
               
                   
                   
                   
                   
                 [Q28243_CANFA] 
               
               
                   
               
               
                   
                 Sequence 
                 m/z [Da] 
                 MH+ [Da] 
                 RT [min] 
               
               
                   
               
               
                   
                 IMGSDSDIFTNIGTP 
                  945.45618 
                 3778.80288 
                   26.50 
                   
                   
               
               
                   
                 EFPSSGKTSSHSKQF 
                   
                   
                   
                   
                   
               
               
                   
                 VTSSTT 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 4) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 THIMGSDSDIFTNIG 
                  738.34826 
                 2950.37119 
                   26.38 
                   
                   
               
               
                   
                 TPEFPSSGKTSSH 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 5) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 THIMGSDSDIFTNIG 
                 1013.13733 
                 3037.39743 
                   26.25 
                   
                   
               
               
                   
                 TPEFPSSGKTSSHS 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 6) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 IMGSDSDIFTNIGTP 
                  933.76956 
                 2799.29412 
                   27.29 
                   
                   
               
               
                   
                 EFPSSGKTSSHS 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 7) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 P12278 
                  6 
                  101 
                   11.2 
                 Apolipoprotein C- 
                   
                 Increased 
               
               
                   
                   
                   
                   
                 II OS =  Canis   
                   
                   
               
               
                   
                   
                   
                   
                 
                   familiaris 
                 
                   
                   
               
               
                   
                   
                   
                   
                 GN = APOC2 PE = 2 
                   
                   
               
               
                   
                   
                   
                   
                 SV = 1- 
                   
                   
               
               
                   
                   
                   
                   
                 [APOC2_CANFA] 
               
               
                   
               
               
                   
                 Sequence 
                 m/z [Da] 
                 MH+ [Da] 
                 RT [min] 
               
               
                   
               
               
                   
                 AHESQQDETTSSALL 
                 1335.61365 
                 4004.82639 
                   35.25 
                   
                   
               
               
                   
                 TQMQESLYSWGTARS 
                   
                   
                   
                   
                   
               
               
                   
                 AAEDL 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 8) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 AHESQQDETTSSALL 
                 1217.55859 
                 2434.10991 
                   28.22 
                   
                   
               
               
                   
                 TQMQESL 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 9) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 AHESQQDETTSSALL 
                 1088.15869 
                 3262.46152 
                   33.89 
                   
                   
               
               
                   
                 TQMQESLYSWGTA 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 10) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 AHESQQDETTSSALL 
                  812.04224 
                 2434.11216 
                   28.24 
                   
                   
               
               
                   
                 TQMQESL 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 11) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 AHESQQDETTSSALL 
                 1631.73376 
                 3262.46025 
                   33.91 
                   
                   
               
               
                   
                 TQMQESLYSWGTA 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 12) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 AHESQQDETTSSALL 
                  808.87756 
                 1616.74785 
                   20.77 
                   
                   
               
               
                   
                 (SEQ. ID NO: 13) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 P68213 
                  7 
                   28 
                    3.0 
                 Fibrinogen alpha 
                   
                 Decreased 
               
               
                   
                   
                   
                   
                 chain (Fragment) 
                   
                   
               
               
                   
                   
                   
                   
                 OS =  Canis   
                   
                   
               
               
                   
                   
                   
                   
                 
                   familiaris 
                 
                   
                   
               
               
                   
                   
                   
                   
                 GN = FGA PE = 1 
                   
                   
               
               
                   
                   
                   
                   
                 SV = 1- 
                   
                   
               
               
                   
                   
                   
                   
                 [FIBA_CANFA] 
               
               
                   
               
               
                   
                 Sequence 
                 m/z [Da] 
                 MH+ [Da] 
                 RT [min] 
               
               
                   
               
               
                   
                 NSKEGEFIAEGGGV 
                  697.33575 
                 1393.66423 
                   21.26 
                   
                   
               
               
                   
                 (SEQ. ID NO: 14) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 SKEGEFIAEGGGV 
                  640.31421 
                 1279.62114 
                   21.32 
                   
                   
               
               
                   
                 (SEQ. ID NO: 15) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 TNSKEGEFIAEGGGV 
                  747.85939 
                 1494.71151 
                   21.25 
                   
                   
               
               
                   
                 (SEQ. ID NO: 16) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 KEGEFIAEGGGV 
                  596.79858 
                 1192.58989 
                   21.20 
                   
                   
               
               
                   
                 (SEQ. ID NO: 17) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 EGEFIAEGGGV 
                 1064.49362 
                 1064.49362 
                   22.99 
                   
                   
               
               
                   
                 (SEQ. ID NO: 18) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 GEFIAEGGGV 
                  935.44861 
                  935.44861 
                   22.73 
                   
                   
               
               
                   
                 (SEQ. ID NO: 19) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 FIAEGGGV 
                  749.38739 
                  749.38739 
                   20.88 
                   
                   
               
               
                   
                 (SEQ. ID NO: 20) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 XP_535836 
                  4 
                  653 
                   73.1 
                 Kininogen 
                   
                 Decreased 
               
               
                   
               
               
                   
                 Sequence 
                 Charge 
                 m/z [Da] 
                 MH+ [Da] 
                 RT [min] 
               
               
                   
               
               
                   
                 HGGQRELDFDLEHQ 
                    3 
                  560.93286 
                 1680.78403 
                 20.94 
                   
               
               
                   
                 (SEQ. ID NO: 21) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 DEEWDSGKEQGPTHG 
                    3 
                  622.59778 
                 1865.77878 
                 15.61 
                   
               
               
                   
                 HG 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 22) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 ELDFDLEHQ 
                    2 
                  573.26135 
                 1145.51543 
                 24.10 
                   
               
               
                   
                 (SEQ. ID NO: 23) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 DCDYKESTQAATGEC 
                    3 
                  540.87445 
                 1620.60880 
                 26.40 
                   
               
               
                   
                 (SEQ. ID NO: 24) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 XP_848765 &amp; 
                  4 
                  958 
                  105.0 
                 Inter-Alpha 
                   
                 Differ- 
               
               
                 XP_843672 
                   
                   
                   
                 Inhibitor H4 
                   
                 entially 
               
               
                   
                   
                   
                   
                 (ITIH4) 
                   
                 expressed 
               
               
                   
               
               
                   
                 Sequence 
                 Charge 
                 m/z [Da] 
                 MH+ [Da] 
                 RT [min] 
               
               
                   
               
               
                   
                 GSEIVVVGKLRDQSP 
                    3 
                  766.10455 
                 2296.29911 
                 24.87 
                   
               
               
                   
                 DVLSAKV 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 25) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 PRDWKPLLVPASPEN 
                    3 
                  645.01086 
                 1933.01804 
                 18.72 
                   
               
               
                   
                 VD 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 26) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 ETLFSMMPGLNMTMD 
                    2 
                 1172.08431 
                 2343.16135 
                 34.46 
                   
               
               
                   
                 KTGLLL 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 27) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 AETVQ 
                    1 
                  547.27649 
                  547.27649 
                 20.09 
                   
               
               
                   
                 (SEQ. ID NO: 28) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 XP_545130 
                 66.23 
                    3 
                   77 
                    8.8 
                 CysA 
                 Differ- 
               
               
                   
                   
                   
                   
                   
                   
                 entially 
               
               
                   
                   
                   
                   
                   
                   
                 expressed 
               
               
                   
               
               
                   
                 Sequence 
                 Charge 
                 m/z [Da] 
                 MH+ [Da] 
                 RT [min] 
               
               
                   
               
               
                   
                 VGDNSYTHLKIFKGL 
                    3 
                  601.00467 
                 1800.99945 
                 26.31 
                   
               
               
                   
                 P 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 29) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 LTLTGYQTDKSKDDE 
                    3 
                  662.33471 
                 1984.98957 
                 18.10 
                   
               
               
                   
                 LTG 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 30) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 KPQLEEKTNETYQEF 
                    3 
                  695.32800 
                 2083.96946 
                 19.15 
                   
               
               
                   
                 EA 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 31) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 XP_535601 
                 75.32 
                    7 
                   77 
                    9.0 
                 CysB 
                 Decreased 
               
               
                   
               
               
                   
                 Sequence 
                 Charge 
                 m/z [Da] 
                 MH+ [Da] 
                 RT [min] 
               
               
                   
               
               
                   
                 YQTNKAKHDELAYF 
                    3 
                  576.61572 
                 1727.83261 
                 21.46 
                   
               
               
                   
                 (SEQ. ID NO: 32) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 QTNKAKHDELAYF 
                    3 
                  522.26111 
                 1564.76877 
                 20.82 
                   
               
               
                   
                 (SEQ. ID NO: 33) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 ENKPLALSSYQTNK 
                    2 
                  796.91620 
                 1592.82513 
                 27.77 
                   
               
               
                   
                 (SEQ. ID NO: 34) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 QVVAGTPY 
                    1 
                  834.43532 
                  834.43532 
                 31.89 
                   
               
               
                   
                 (SEQ. ID NO: 35) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 EERENKKYTTFK 
                    2 
                  786.90753 
                 1572.80779 
                 31.24 
                   
               
               
                   
                 (SEQ. ID NO: 36) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 YFIKVQVDDDEFVHL 
                    3 
                  675.00958 
                 2023.01419 
                 23.40 
                   
               
               
                   
                 R 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 37) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 VVAGTPYFIKVQVDD 
                    3 
                  589.30709 
                 1765.90671 
                 19.41 
                   
               
               
                   
                 D 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 38) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NP_001013443 
                 43.66 
                   21 
                  568 
                   57.6 
                 Keratin 
                 Differ- 
               
               
                   
                   
                   
                   
                   
                 Type I 
                 entially 
               
               
                   
                   
                   
                   
                   
                 Cyto- 
                 expressed 
               
               
                   
                   
                   
                   
                   
                 skeletal 
                   
               
               
                   
                   
                   
                   
                   
                 10 
               
               
                   
               
               
                   
                 Sequence 
                 Charge 
                 m/z [Da] 
                 MH+ [Da] 
                 RT [min] 
               
               
                   
               
               
                   
                 MQNLNDRLAS 
                    2 
                  581.28491 
                 1161.56255 
                 20.90 
                   
               
               
                   
                 (SEQ. ID NO: 39) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 FGGGYGGVSFGGGSF 
                    3 
                  624.60724 
                 1871.80716 
                 19.91 
                   
               
               
                   
                 GGGSFGG 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 40) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 SFGGGYGGVSFG 
                    2 
                  546.24731 
                 1091.48735 
                 25.33 
                   
               
               
                   
                 (SEQ. ID NO: 41) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 FSRGSSGGGCFGGSS 
                    3 
                  656.61829 
                 1967.84031 
                 28.01 
                   
               
               
                   
                 GGYGGLGG 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 42) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 EEQLQ 
                    1 
                  646.30862 
                  646.30862 
                 15.60 
                   
               
               
                   
                 (SEQ. ID NO: 43) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 QNRKDAEAWFNEKSK 
                    3 
                  617.64661 
                 1850.92527 
                 19.80 
                   
               
               
                   
                 (SEQ. ID NO: 44) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 PRDYSKYYQTIEDLK 
                    3 
                  758.71680 
                 2274.13584 
                 26.49 
                   
               
               
                   
                 NQI 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 45) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 KDAEAWFNEKSKEL 
                    3 
                  565.61548 
                 1694.83188 
                 19.42 
                   
               
               
                   
                 (SEQ. ID NO: 46) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 KYENEVALRQSVEA 
                    3 
                  545.94529 
                 1635.82131 
                 19.39 
                   
               
               
                   
                 (SEQ. ID NO: 47) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 KSKELTTEINSNIEQ 
                    3 
                  622.31818 
                 1864.93998 
                 19.60 
                   
               
               
                   
                 M 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 48) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 LQIDN 
                    1 
                  602.31784 
                  602.31784 
                 16.07 
                   
               
               
                   
                 (SEQ. ID NO: 49) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 SIGGGFSSGG 
                    1 
                  825.37653 
                  825.37653 
                 34.30 
                   
               
               
                   
                 (SEQ. ID NO: 50) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 FGGGGFSGGSFGGYG 
                    3 
                  719.64934 
                 2156.93346 
                 23.14 
                   
               
               
                   
                 GGYGGDGGLL 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 51) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 LENEIQTYRSLLEGE 
                    3 
                  617.64661 
                 1850.92527 
                 19.80 
                   
               
               
                   
                 G 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 52) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 GSIGGGFSSG 
                    1 
                  825.37653 
                  825.37653 
                 34.30 
                   
               
               
                   
                 (SEQ. ID NO: 53) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 EDLKNQILNLTTDN 
                    2 
                  815.92169 
                 1630.83611 
                 26.45 
                   
               
               
                   
                 (SEQ. ID NO: 54) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 GGGGYGGGSSGGGGS 
                    3 
                  537.21368 
                 1609.62650 
                 19.61 
                   
               
               
                   
                 HGGSSGG 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 55) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 GRYCVQLSQIQAQIS 
                    2 
                  890.94928 
                 1780.89128 
                 20.25 
                   
               
               
                   
                 S 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 56) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 RVLDELTLT 
                    1 
                 1059.60266 
                 1059.60266 
                 33.79 
                   
               
               
                   
                 (SEQ. ID NO: 57) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 RLASYLDKVRALEES 
                    2 
                 1014.02356 
                 2027.03984 
                 37.86 
                   
               
               
                   
                 NY 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 58) 
                   
                   
                   
                   
                   
               
               
                   
               
               
                   
                 GGGYGGDGGLLSGNE 
                    2 
                  768.86627 
                 1536.72527 
                 22.51 
                   
               
               
                   
                 KV 
                   
                   
                   
                   
                   
               
               
                   
                 (SEQ. ID NO: 59) 
               
               
                   
               
            
           
         
       
     
     Although methods for performing LC/MS are well known in the art, the specific liquid chromatography/mass spectrometry methods utilized for the present study are provided below: 
     Liquid Chromatography Parameters 
     Solvent A: 0.1% Formic acid in water; Solvent B: 0.1% Formic acid in acetonitrile; Column: Acquity UPLC BEH130 C18 1.7 μM 2.1id×150 mm length; Guard Column: vanguard BEH 300 C 18  1.7 uM; Injection volume: 25 μL; Tray temp: 10° C.; Column oven temp: 45° C.; MS run time: 60 minutes; Divert valve: none 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Gradient for 35% fraction 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 No 
                 Time 
                 A % 
                 B % 
                 C % 
                 D % 
                 μL/min 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 1 
                 0 
                 100 
                 0 
                 0 
                 0 
                 300 
               
               
                   
                 2 
                 5 
                 100 
                 0 
                 0 
                 0 
                 300 
               
               
                   
                 3 
                 45 
                 50 
                 50 
                 0 
                 0 
                 300 
               
               
                   
                 4 
                 46 
                 100 
                 0 
                 0 
                 0 
                 300 
               
               
                   
                 5 
                 60 
                 100 
                 0 
                 0 
                 0 
                 300 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Gradient for 65% fraction 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 No 
                 Time 
                 A % 
                 B % 
                 C % 
                 D % 
                 μL/min 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 1 
                 0 
                 70 
                 30 
                 0 
                 0 
                 300 
               
               
                   
                 2 
                 5 
                 70 
                 30 
                 0 
                 0 
                 300 
               
               
                   
                 3 
                 45 
                 25 
                 75 
                 0 
                 0 
                 300 
               
               
                   
                 4 
                 46 
                 70 
                 30 
                 0 
                 0 
                 300 
               
               
                   
                 5 
                 60 
                 70 
                 30 
                 0 
                 0 
                 300 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                   
               
               
                 Mass Spectrometry Parameters and Methods 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 MS scan event 1: FTMS; resolution 30000; scan range 500.0-2000.0 
               
               
                 MS scan event 2-6: ITMS + c norm Dep MS/MS 1 st , 2 nd , 3 rd  most 
               
               
                 intense ion from scan 1 for differential expression and from list for 
               
               
                 targeted identification 
               
               
                  Activation Type: CID 
               
               
                  Min Signal Required: 500 
               
               
                  Isolation Width: 1.5 
               
               
                  Normalized Coll. Energy: 35.0 
               
               
                  Default Charge State: 2 
               
               
                  Activation Q: 0.250 
               
               
                  Activation Time: 30.000 
               
               
                  CV = 0.0 V 
               
               
                 Data Dependent Settings for differential expression: 
               
               
                  Use separate polarity settings disabled 
               
               
                  Parent Mass List: none 
               
               
                  Reject Mass List: none 
               
               
                  Neutral loss Mass List: none 
               
               
                  Product Mass List: none 
               
               
                  Neutral loss in top: 3 
               
               
                  Product in top: 3 
               
               
                  Most intense if no parent masses found not enabled 
               
               
                  Add/subtract mass not enabled 
               
               
                  FT master scan preview mode enabled 
               
               
                  Charge state screening enabled 
               
               
                  Monoisotopic precursor selection enabled 
               
               
                  Non-peptide monoisotopic recognition not enabled 
               
               
                  Charge state rejection enabled 
               
               
                   Unassigned charge states: rejected 
               
               
                   Charge state 1: not rejected 
               
               
                   Charge state 2: not rejected 
               
               
                   Charge state 3: not rejected 
               
               
                   Charge state 4+: not rejected 
               
               
                 Data Dependent Settings for targeted identification: 
               
               
                  Use separate polarity settings disabled 
               
               
                  Reject Mass List: none 
               
               
                  Neutral loss Mass List: none 
               
               
                  Product Mass List: none 
               
               
                  Neutral loss in top: 3 
               
               
                  Product in top: 3 
               
               
                  Most intense if no parent masses found not enabled 
               
               
                  Add/subtract mass not enabled 
               
               
                  FT master scan preview mode enabled 
               
               
                  Charge state screening enabled 
               
               
                  Monoisotopic precursor selection enabled 
               
               
                  Non-peptide monoisotopic recognition not enabled 
               
               
                  Charge state rejection enabled 
               
               
                   Unassigned charge states: rejected 
               
               
                   Charge state 1: not rejected 
               
               
                   Charge state 2: not rejected 
               
               
                   Charge state 3: not rejected 
               
               
                   Charge state 4+: not rejected 
               
               
                 Global Data Dependent Settings” 
               
               
                  Use global parent and reject mass lists not enabled for 
               
               
                  differential expression and enabled for targeted identification 
               
               
                  Exclude parent mass from data dependent selection not 
               
               
                 enabled 
               
               
                  Exclusion mass width relative to mass 
               
               
                  Exclusion mass width relative to low (ppm): 20.00 
               
               
                  Exclusion mass width relative to high (ppm): 20.00 
               
               
                  Parent mass width relative to mass 
               
               
                  Parent mass width low (ppm): 10.00 
               
               
                  Parent mass width high (ppm): 10.00 
               
               
                  Reject mass width relative to mass 
               
               
                  Reject mass width low (ppm): 20.00 
               
               
                  Reject mass width high (ppm): 20.00 
               
               
                  Zoom/UltraZoom scan mass width by mass 
               
               
                  Zoom/UltraZoom scan mass low: 5.00 
               
               
                  Zoom/UltraZoom scan mass high: 5.00 
               
               
                  FT SIM scan mass width low: 5.00 
               
               
                  FT SIM scan mass width high: 5.00 
               
               
                  Neutral Loss candidates processed by decreasing intensity 
               
               
                  Neutral loss mass width by mass 
               
               
                  Neutral Loss mass width low: 0.50000 
               
               
                  Neutral Loss mass width high: 0.50000 
               
               
                  Product candidates processed by decreasing intensity 
               
               
                  Product mass width by mass 
               
               
                  Product mass width low: 0.50000 
               
               
                  Product mass width high: 0.50000 
               
               
                  MS mass range: 0.00-1000000.00 
               
               
                  Use m/z values as masses not enabled 
               
               
                  Analog UV data dep. Not enabled 
               
               
                  Dynamic exclusion enabled 
               
               
                   Repeat Count: 2 
               
               
                   Repeat Duration: 30.00 
               
               
                   Exclusion List Size: 500 
               
               
                   Exclusion Duration: 60.00 
               
               
                   Exclusion mass width relative to mass 
               
               
                   Exclusion mass width low (ppm): 20.00 
               
               
                   Exclusion mass width high (ppm): 20.00 
               
               
                  Isotopic data dependence not enabled 
               
               
                  Mass Tags data dependence not enabled 
               
               
                  Custom Data Dependent Settings not enabled 
               
               
                 MS Tune File Values 
               
               
                 Source Type: ESI 
               
               
                 Capillary Temp (° C.): 250.00 
               
               
                 Sheath gas Flow: 24.00 
               
               
                 Aux Gas Flow: 13.00 
               
               
                 Sweep Gas Flow: 0 
               
               
                 Ion Trap MSn AGC Target: 10000 
               
               
                 FTMS Injection waveforms: off 
               
               
                 FTMS AGC Target: 500000 
               
               
                 Source voltage (kV): 4.50 
               
               
                 Source current (μA): 100.00 
               
               
                 Capillary Voltage (V): 68.28 
               
               
                 Tube Lens (V): 130.00 
               
               
                 Skimmer Offset (V): 0.00 
               
               
                 Multipole RF Amplifier (Vp-p): 550.00 
               
               
                 Multipole 00 offset (V): −1.60 
               
               
                 Lens 0 Voltage (V): −2.70 
               
               
                 Multipole 0 offset (V): −5.80 
               
               
                 Lens 1 Voltage (V): −11.00 
               
               
                 Gate Lens offset (V): −60.00 
               
               
                 Multipole 1 offset (V): −10.50 
               
               
                 Front Lens (V): −5.18 
               
               
                 FTMS full microscans: 1 
               
               
                 FTMS full Max Ion Time (ms): 500 
               
               
                 Ion Trap MSn Micro Scans: 3 
               
               
                 Ion Trap MSn Max Ion Time: 100 
               
               
                   
               
            
           
         
       
     
     The mass spec data from the above analysis were analyzed for differential expression of the peptides using SIEVE 1.3 software with the following parameters: 
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 SIEVE Parameters 
               
               
                   
               
             
            
               
                 Alignment Parameters 
               
            
           
           
               
               
               
            
               
                   
                 AlignmentBypass 
                 False 
               
               
                   
                 CorrelationBinWidth 
                 1 
               
               
                   
                 RT LimitsForAlignment 
                 True 
               
               
                   
                 TileIncrement 
                 150 
               
               
                   
                 TileMaximum 
                 300 
               
               
                   
                 TileSize 
                 300 
               
               
                   
                 Tile Threshold 
                 0.6 
               
            
           
           
               
            
               
                 Analysis Definition 
               
            
           
           
               
               
               
            
               
                   
                 Experiment Target 
                 PROTEOMICS 
               
               
                   
                 Experiment Type 
                 AVSB 
               
            
           
           
               
            
               
                 Frame Parameters 
               
            
           
           
               
               
               
            
               
                   
                 AvgChargeProcessor 
                 False 
               
               
                   
                 ControlGroup 
                 c 
               
               
                   
                 FramelDCriteria 
                 ORDER BY PVALUE ASC 
               
               
                   
                 FrameSeedFile 
                   
               
               
                   
                 KMClusters 
                 10 
               
               
                   
                 MS2CorrProcessor 
                 False 
               
               
                   
                 MZStart 
                 500 
               
               
                   
                 MZStop 
                 2000 
               
               
                   
                 MZWidth 
                 0.01 
               
               
                   
                 ProcessorModules 
                 PCA V1.0:ROC V1.0 
               
               
                   
                 RTStart 
                 0 
               
               
                   
                 RTStop 
                 59.98 
               
               
                   
                 RTWidth 
                 1.5 
               
               
                   
                 UseTICNormalizedRatios 
                 False 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Global Parent mass 35% fraction 
               
               
                 for targeted identification: 
               
            
           
           
               
               
               
            
               
                   
                 start time 
                 End time 
               
               
                 m/z 
                 (min) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 500.837 
                 20.8 
                 21.4 
               
               
                 511.557 
                 20.8 
                 21.4 
               
               
                 516.216 
                 20.8 
                 21.4 
               
               
                 529.195 
                 20.8 
                 21.4 
               
               
                 534.519 
                 20.8 
                 21.4 
               
               
                 540.878 
                 23.4 
                 24.0 
               
               
                 549.959 
                 44.1 
                 44.7 
               
               
                 554.519 
                 22.1 
                 22.7 
               
               
                 586.686 
                 23.3 
                 23.9 
               
               
                 588.915 
                 20.8 
                 21.4 
               
               
                 590.986 
                 44.1 
                 44.7 
               
               
                 596.798 
                 20.7 
                 21.3 
               
               
                 630.336 
                 26.0 
                 26.6 
               
               
                 632.392 
                 37.0 
                 37.6 
               
               
                 640.314 
                 20.8 
                 21.4 
               
               
                 646.067 
                 17.9 
                 18.5 
               
               
                 661.491 
                 35.0 
                 35.6 
               
               
                 662.294 
                 27.1 
                 27.7 
               
               
                 666.330 
                 37.3 
                 37.9 
               
               
                 666.770 
                 20.8 
                 21.4 
               
               
                 697.336 
                 20.7 
                 21.3 
               
               
                 697.837 
                 20.7 
                 21.3 
               
               
                 714.396 
                 27.1 
                 27.7 
               
               
                 722.599 
                 20.7 
                 21.3 
               
               
                 732.085 
                 20.7 
                 21.3 
               
               
                 736.079 
                 20.7 
                 21.3 
               
               
                 745.375 
                 28.8 
                 29.4 
               
               
                 747.859 
                 20.7 
                 21.3 
               
               
                 748.299 
                 20.8 
                 21.4 
               
               
                 746.279 
                 22.5 
                 23.1 
               
               
                 758.347 
                 20.8 
                 21.4 
               
               
                 761.089 
                 20.8 
                 21.4 
               
               
                 762.952 
                 33.1 
                 33.7 
               
               
                 766.832 
                 20.8 
                 21.4 
               
               
                 770.824 
                 20.8 
                 21.4 
               
               
                 774.316 
                 20.8 
                 21.4 
               
               
                 785.495 
                 37.0 
                 37.6 
               
               
                 792.484 
                 37.0 
                 37.6 
               
               
                 798.662 
                 27.1 
                 27.7 
               
               
                 815.324 
                 23.7 
                 24.3 
               
               
                 815.292 
                 22.0 
                 22.6 
               
               
                 831.276 
                 22.0 
                 22.6 
               
               
                 845.270 
                 27.1 
                 27.7 
               
               
                 857.071 
                 27.1 
                 27.7 
               
               
                 883.346 
                 36.5 
                 37.1 
               
               
                 888.005 
                 20.8 
                 21.4 
               
               
                 908.015 
                 20.8 
                 21.4 
               
               
                 926.783 
                 21.2 
                 21.8 
               
               
                 929.445 
                 20.7 
                 21.3 
               
               
                 946.081 
                 37.0 
                 37.6 
               
               
                 963.128 
                 20.7 
                 21.3 
               
               
                 972.536 
                 37.0 
                 37.6 
               
               
                 980.768 
                 20.7 
                 21.3 
               
               
                 996.811 
                 20.7 
                 21.3 
               
               
                 999.409 
                 20.7 
                 21.3 
               
               
                 1014.449 
                 20.8 
                 21.4 
               
               
                 1017.377 
                 39.7 
                 40.3 
               
               
                 1017.250 
                 39.6 
                 40.2 
               
               
                 1034.163 
                 36.5 
                 37.1 
               
               
                 1061.032 
                 38.0 
                 38.6 
               
               
                 1071.011 
                 20.7 
                 21.3 
               
               
                 1073.287 
                 38.3 
                 38.9 
               
               
                 1074.429 
                 43.6 
                 44.2 
               
               
                 1075.546 
                 43.0 
                 43.6 
               
               
                 1078.177 
                 40.3 
                 40.9 
               
               
                 1083.736 
                 22.1 
                 22.7 
               
               
                 1089.401 
                 38.0 
                 38.6 
               
               
                 1096.026 
                 20.7 
                 21.3 
               
               
                 1101.960 
                 43.0 
                 43.6 
               
               
                 1104.411 
                 37.4 
                 38.0 
               
               
                 1109.504 
                 20.7 
                 21.3 
               
               
                 1117.566 
                 42.9 
                 43.5 
               
               
                 1141.310 
                 40.2 
                 40.8 
               
               
                 1140.059 
                 40.2 
                 40.8 
               
               
                 1162.715 
                 39.6 
                 40.2 
               
               
                 1162.285 
                 39.6 
                 40.2 
               
               
                 1175.385 
                 42.8 
                 43.4 
               
               
                 1175.963 
                 20.7 
                 21.3 
               
               
                 1182.040 
                 36.5 
                 37.1 
               
               
                 1185.065 
                 38.6 
                 39.2 
               
               
                 1184.315 
                 38.7 
                 39.3 
               
               
                 1184.044 
                 36.5 
                 37.1 
               
               
                 1186.945 
                 20.7 
                 21.3 
               
               
                 1189.456 
                 36.5 
                 37.1 
               
               
                 1197.564 
                 38.3 
                 38.9 
               
               
                 1201.052 
                 38.0 
                 38.6 
               
               
                 1205.551 
                 27.1 
                 27.7 
               
               
                 1221.832 
                 38.1 
                 38.7 
               
               
                 1221.330 
                 38.0 
                 38.6 
               
               
                 1221.956 
                 38.1 
                 38.7 
               
               
                 1221.203 
                 38.0 
                 38.6 
               
               
                 1229.059 
                 41.7 
                 42.3 
               
               
                 1229.337 
                 43.0 
                 43.6 
               
               
                 1234.050 
                 43.3 
                 43.9 
               
               
                 1234.184 
                 43.2 
                 43.8 
               
               
                 1237.197 
                 38.1 
                 38.7 
               
               
                 1239.714 
                 38.3 
                 38.9 
               
               
                 1239.903 
                 40.3 
                 40.9 
               
               
                 1239.906 
                 22.0 
                 22.6 
               
               
                 1239.336 
                 39.3 
                 39.9 
               
               
                 1241.893 
                 22.0 
                 22.6 
               
               
                 1245.780 
                 42.9 
                 43.5 
               
               
                 1244.893 
                 41.7 
                 42.3 
               
               
                 1244.821 
                 38.2 
                 38.8 
               
               
                 1252.485 
                 42.9 
                 43.5 
               
               
                 1253.180 
                 38.0 
                 38.6 
               
               
                 1262.757 
                 41.7 
                 42.3 
               
               
                 1269.226 
                 43.0 
                 43.6 
               
               
                 1268.942 
                 42.9 
                 43.5 
               
               
                 1271.795 
                 41.7 
                 42.3 
               
               
                 1271.940 
                 41.7 
                 42.3 
               
               
                 1272.223 
                 41.7 
                 42.3 
               
               
                 1271.511 
                 41.7 
                 42.3 
               
               
                 1271.366 
                 41.5 
                 42.1 
               
               
                 1271.653 
                 43.6 
                 44.2 
               
               
                 1271.939 
                 42.9 
                 43.5 
               
               
                 1271.663 
                 43.0 
                 43.6 
               
               
                 1279.356 
                 41.7 
                 42.3 
               
               
                 1279.640 
                 41.7 
                 42.3 
               
               
                 1280.487 
                 43.1 
                 43.7 
               
               
                 1282.341 
                 42.9 
                 43.5 
               
               
                 1282.623 
                 42.9 
                 43.5 
               
               
                 1283.501 
                 41.7 
                 42.3 
               
               
                 1283.215 
                 41.7 
                 42.3 
               
               
                 1287.661 
                 42.8 
                 43.4 
               
               
                 1287.380 
                 42.8 
                 43.4 
               
               
                 1287.671 
                 42.9 
                 43.5 
               
               
                 1289.949 
                 41.4 
                 42.0 
               
               
                 1290.093 
                 41.4 
                 42.0 
               
               
                 1290.231 
                 41.4 
                 42.0 
               
               
                 1295.514 
                 42.8 
                 43.4 
               
               
                 1302.780 
                 40.2 
                 40.8 
               
               
                 1313.671 
                 42.8 
                 43.4 
               
               
                 1326.085 
                 42.9 
                 43.5 
               
               
                 1329.279 
                 41.4 
                 42.0 
               
               
                 1340.257 
                 42.8 
                 43.4 
               
               
                 1353.504 
                 38.6 
                 39.2 
               
               
                 1353.226 
                 38.0 
                 38.6 
               
               
                 1354.345 
                 38.0 
                 38.6 
               
               
                 1355.998 
                 39.6 
                 40.2 
               
               
                 1361.344 
                 38.6 
                 39.2 
               
               
                 1378.833 
                 43.0 
                 43.6 
               
               
                 1393.662 
                 20.7 
                 21.3 
               
               
                 1395.947 
                 38.0 
                 38.6 
               
               
                 1395.808 
                 38.1 
                 38.7 
               
               
                 1396.095 
                 38.0 
                 38.6 
               
               
                 1396.238 
                 38.1 
                 38.7 
               
               
                 1403.093 
                 38.1 
                 38.7 
               
               
                 1405.390 
                 37.0 
                 37.6 
               
               
                 1412.808 
                 42.9 
                 43.5 
               
               
                 1416.957 
                 38.2 
                 38.8 
               
               
                 1417.242 
                 38.2 
                 38.8 
               
               
                 1416.813 
                 38.2 
                 38.8 
               
               
                 1415.521 
                 38.3 
                 38.9 
               
               
                 1416.365 
                 38.2 
                 38.8 
               
               
                 1423.711 
                 43.0 
                 43.6 
               
               
                 1432.054 
                 43.1 
                 43.7 
               
               
                 1434.534 
                 38.3 
                 38.9 
               
               
                 1444.691 
                 20.7 
                 21.3 
               
               
                 1461.219 
                 39.3 
                 39.9 
               
               
                 1466.392 
                 38.3 
                 38.9 
               
               
                 1480.934 
                 43.0 
                 43.6 
               
               
                 1480.763 
                 42.9 
                 43.5 
               
               
                 1483.434 
                 41.5 
                 42.1 
               
               
                 1483.261 
                 41.5 
                 42.1 
               
               
                 1483.594 
                 43.1 
                 43.7 
               
               
                 1483.096 
                 41.5 
                 42.1 
               
               
                 1488.084 
                 41.5 
                 42.1 
               
               
                 1488.253 
                 41.5 
                 42.1 
               
               
                 1492.580 
                 41.8 
                 42.4 
               
               
                 1492.738 
                 43.1 
                 43.7 
               
               
                 1494.710 
                 20.7 
                 21.3 
               
               
                 1493.188 
                 41.4 
                 42.0 
               
               
                 1501.262 
                 43.1 
                 43.7 
               
               
                 1501.243 
                 42.8 
                 43.4 
               
               
                 1504.607 
                 41.4 
                 42.0 
               
               
                 1519.408 
                 40.2 
                 40.8 
               
               
                 1519.963 
                 42.8 
                 43.4 
               
               
                 1533.454 
                 41.4 
                 42.0 
               
               
                 1550.589 
                 42.9 
                 43.5 
               
               
                 1567.262 
                 43.0 
                 43.6 
               
               
                 1566.964 
                 43.2 
                 43.8 
               
               
                 1616.809 
                 42.8 
                 43.4 
               
               
                 1625.276 
                 38.1 
                 38.7 
               
               
                 1682.802 
                 41.4 
                 42.0 
               
               
                 1708.700 
                 35.2 
                 35.8 
               
               
                 1715.693 
                 43.0 
                 43.6 
               
               
                 1719.490 
                 42.8 
                 43.4 
               
               
                 1720.363 
                 43.1 
                 43.7 
               
               
                 1720.076 
                 41.7 
                 42.3 
               
               
                 1735.925 
                 42.9 
                 43.5 
               
               
                 1735.448 
                 42.9 
                 43.5 
               
               
                 1742.423 
                 42.8 
                 43.4 
               
               
                 1742.691 
                 39.3 
                 39.9 
               
               
                 1749.090 
                 42.8 
                 43.4 
               
               
                 1755.091 
                 42.9 
                 43.5 
               
               
                 1766.811 
                 43.0 
                 43.6 
               
               
                 1769.294 
                 42.9 
                 43.5 
               
               
                 1775.798 
                 43.0 
                 43.6 
               
               
                 1802.490 
                 42.8 
                 43.4 
               
               
                 1808.484 
                 42.9 
                 43.5 
               
               
                 1822.120 
                 41.4 
                 42.0 
               
               
                 1893.263 
                 5.0 
                 60.0 
               
               
                 1796.466 
                 5.0 
                 60.0 
               
               
                 1596.971 
                 5.0 
                 60.0 
               
               
                 1368.976 
                 5.0 
                 60.0 
               
               
                 1150.101 
                 5.0 
                 60.0 
               
               
                 1635.848 
                 5.0 
                 60.0 
               
               
                 1338.604 
                 5.0 
                 60.0 
               
               
                 921.201 
                 5.0 
                 60.0 
               
               
                 775.405 
                 5.0 
                 60.0 
               
               
                 1618.973 
                 5.0 
                 60.0 
               
               
                 1324.797 
                 5.0 
                 60.0 
               
               
                 1121.137 
                 5.0 
                 60.0 
               
               
                 911.113 
                 5.0 
                 60.0 
               
               
                 809.990 
                 5.0 
                 60.0 
               
               
                 1529.751 
                 5.0 
                 60.0 
               
               
                 1384.157 
                 5.0 
                 60.0 
               
               
                 1263.883 
                 5.0 
                 60.0 
               
               
                 1211.263 
                 5.0 
                 60.0 
               
               
                 1162.853 
                 5.0 
                 60.0 
               
               
                 1247.480 
                 5.0 
                 60.0 
               
               
                 1366.192 
                 5.0 
                 60.0 
               
               
                 1510.899 
                 5.0 
                 60.0 
               
               
                 1950.616 
                 5.0 
                 60.0 
               
               
                 1540.172 
                 5.0 
                 60.0 
               
               
                 1170.773 
                 5.0 
                 60.0 
               
               
                 1090.293 
                 5.0 
                 60.0 
               
               
                 1185.014 
                 5.0 
                 60.0 
               
               
                 1362.615 
                 5.0 
                 60.0 
               
               
                 1542.070 
                 5.0 
                 60.0 
               
               
                 1445.754 
                 5.0 
                 60.0 
               
               
                 1360.769 
                 5.0 
                 60.0 
               
               
                 1285.227 
                 5.0 
                 60.0 
               
               
                 1217.636 
                 5.0 
                 60.0 
               
               
                 1156.805 
                 5.0 
                 60.0 
               
               
                 1101.767 
                 5.0 
                 60.0 
               
               
                 1051.732 
                 5.0 
                 60.0 
               
               
                 1006.048 
                 5.0 
                 60.0 
               
               
                 964.172 
                 5.0 
                 60.0 
               
               
                 14138.386 
                 5.0 
                 60.0 
               
               
                 1768.180 
                 5.0 
                 60.0 
               
               
                 1286.224 
                 5.0 
                 60.0 
               
               
                 1088.498 
                 5.0 
                 60.0 
               
               
                 943.499 
                 5.0 
                 60.0 
               
               
                 884.593 
                 5.0 
                 60.0 
               
               
                 786.924 
                 5.0 
                 60.0 
               
               
                 745.080 
                 5.0 
                 60.0 
               
               
                 954.251 
                 5.0 
                 60.0 
               
               
                 1040.909 
                 5.0 
                 60.0 
               
               
                 1144.899 
                 5.0 
                 60.0 
               
               
                 1205.104 
                 5.0 
                 60.0 
               
               
                 1526.197 
                 5.0 
                 60.0 
               
               
                 1430.872 
                 5.0 
                 60.0 
               
               
                 1907.494 
                 5.0 
                 60.0 
               
               
                 1760.841 
                 5.0 
                 60.0 
               
               
                 1977.132 
                 5.0 
                 60.0 
               
               
                 1757.527 
                 5.0 
                 60.0 
               
               
                 1581.907 
                 5.0 
                 60.0 
               
               
                 1438.189 
                 5.0 
                 60.0 
               
               
                 1054.941 
                 5.0 
                 60.0 
               
               
                 879.285 
                 5.0 
                 60.0 
               
               
                 659.716 
                 5.0 
                 60.0 
               
               
                 1897.745 
                 5.0 
                 60.0 
               
               
                 1660.653 
                 5.0 
                 60.0 
               
               
                 1022.328 
                 5.0 
                 60.0 
               
               
                 633.253 
                 5.0 
                 60.0 
               
               
                 1831.242 
                 5.0 
                 60.0 
               
               
                 1664.857 
                 5.0 
                 60.0 
               
               
                 1526.203 
                 5.0 
                 60.0 
               
               
                 1408.880 
                 5.0 
                 60.0 
               
               
                 1308.318 
                 5.0 
                 60.0 
               
               
                 796.762 
                 5.0 
                 60.0 
               
               
                 733.101 
                 5.0 
                 60.0 
               
               
                 1991.952 
                 5.0 
                 60.0 
               
               
                 1770.739 
                 5.0 
                 60.0 
               
               
                 1593.766 
                 5.0 
                 60.0 
               
               
                 1448.969 
                 5.0 
                 60.0 
               
               
                 1328.306 
                 5.0 
                 60.0 
               
               
                 1226.206 
                 5.0 
                 60.0 
               
               
                 1138.692 
                 5.0 
                 60.0 
               
               
                 1062.846 
                 5.0 
                 60.0 
               
               
                 996.481 
                 5.0 
                 60.0 
               
               
                 937.924 
                 5.0 
                 60.0 
               
               
                 885.873 
                 5.0 
                 60.0 
               
               
                 839.301 
                 5.0 
                 60.0 
               
               
                 797.909 
                 5.0 
                 60.0 
               
               
                 759.464 
                 5.0 
                 60.0 
               
               
                 724.988 
                 5.0 
                 60.0 
               
               
                 693.511 
                 5.0 
                 60.0 
               
               
                 664.657 
                 5.0 
                 60.0 
               
               
                 638.111 
                 5.0 
                 60.0 
               
               
                 1427.610 
                 5.0 
                 60.0 
               
               
                 1223.810 
                 5.0 
                 60.0 
               
               
                 1070.959 
                 5.0 
                 60.0 
               
               
                 856.969 
                 5.0 
                 60.0 
               
               
                 779.154 
                 5.0 
                 60.0 
               
               
                 782.314 
                 5.0 
                 60.0 
               
               
                 626.056 
                 5.0 
                 60.0 
               
               
                 1042.755 
                 5.0 
                 60.0 
               
               
                 1037.510 
                 5.0 
                 60.0 
               
               
                 692.009 
                 5.0 
                 60.0 
               
               
                 519.259 
                 5.0 
                 60.0 
               
               
                 1291.643 
                 5.0 
                 60.0 
               
               
                 861.431 
                 5.0 
                 60.0 
               
               
                 646.325 
                 5.0 
                 60.0 
               
               
                 1480.558 
                 5.0 
                 60.0 
               
               
                 905.177 
                 5.0 
                 60.0 
               
               
                 857.589 
                 5.0 
                 60.0 
               
               
                 1394.901 
                 5.0 
                 60.0 
               
               
                 761.313 
                 5.0 
                 60.0 
               
               
                 1104.010 
                 5.0 
                 60.0 
               
               
                 631.727 
                 5.0 
                 60.0 
               
               
                 883.410 
                 5.0 
                 60.0 
               
               
                 1768.860 
                 5.0 
                 60.0 
               
               
                 708.148 
                 5.0 
                 60.0 
               
               
                 590.291 
                 5.0 
                 60.0 
               
               
                 785.635 
                 5.0 
                 60.0 
               
               
                 845.991 
                 5.0 
                 60.0 
               
               
                 916.407 
                 5.0 
                 60.0 
               
               
                 999.625 
                 5.0 
                 60.0 
               
               
                 1221.540 
                 5.0 
                 60.0 
               
               
                 1831.806 
                 5.0 
                 60.0 
               
               
                 1615.749 
                 5.0 
                 60.0 
               
               
                 1243.116 
                 5.0 
                 60.0 
               
               
                 1077.502 
                 5.0 
                 60.0 
               
               
                 950.855 
                 5.0 
                 60.0 
               
               
                 850.871 
                 5.0 
                 60.0 
               
               
                 1177.032 
                 5.0 
                 60.0 
               
               
                 969.498 
                 5.0 
                 60.0 
               
               
                 1098.630 
                 5.0 
                 60.0 
               
               
                 1862.260 
                 5.0 
                 60.0 
               
               
                 1676.135 
                 5.0 
                 60.0 
               
               
                 1289.567 
                 5.0 
                 60.0 
               
               
                 1117.759 
                 5.0 
                 60.0 
               
               
                 882.653 
                 5.0 
                 60.0 
               
               
                 1480.757 
                 5.0 
                 60.0 
               
               
                 1253.103 
                 5.0 
                 60.0 
               
               
                 1018.335 
                 5.0 
                 60.0 
               
               
                 905.299 
                 5.0 
                 60.0 
               
               
                 1472.097 
                 5.0 
                 60.0 
               
               
                 1104.325 
                 5.0 
                 60.0 
               
               
                 1766.315 
                 5.0 
                 60.0 
               
               
                 883.661 
                 5.0 
                 60.0 
               
               
                 679.972 
                 5.0 
                 60.0 
               
               
                 589.443 
                 5.0 
                 60.0 
               
               
                 1809.590 
                 5.0 
                 60.0 
               
               
                 1357.444 
                 5.0 
                 60.0 
               
               
                 1086.157 
                 5.0 
                 60.0 
               
               
                 958.492 
                 5.0 
                 60.0 
               
               
                 857.704 
                 5.0 
                 60.0 
               
               
                 1286.224 
                 5.0 
                 60.0 
               
               
                 1768.180 
                 5.0 
                 60.0 
               
               
                 1571.827 
                 5.0 
                 60.0 
               
               
                 1414.745 
                 5.0 
                 60.0 
               
               
                 1179.883 
                 5.0 
                 60.0 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                 Start 
                 End 
               
               
                 MZ 
                 Time 
                 Time 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 747.8585 
                 20.963 
                 21.963 
               
               
                 748.3594 
                 20.963 
                 21.963 
               
               
                 1494.711 
                 20.973 
                 21.973 
               
               
                 1393.662 
                 20.925 
                 21.925 
               
               
                 997.1431 
                 20.963 
                 21.963 
               
               
                 1091.809 
                 43.558 
                 44.558 
               
               
                 758.9495 
                 23.687 
                 24.687 
               
               
                 963.4607 
                 20.963 
                 21.963 
               
               
                 996.8089 
                 20.963 
                 21.963 
               
               
                 529.4085 
                 20.079 
                 21.079 
               
               
                 963.1265 
                 20.963 
                 21.963 
               
               
                 1495.694 
                 21.586 
                 22.586 
               
               
                 939.1018 
                 37.446 
                 38.446 
               
               
                 785.4966 
                 37.446 
                 38.446 
               
               
                 1279.621 
                 20.973 
                 21.973 
               
               
                 938.6002 
                 37.446 
                 38.446 
               
               
                 632.3923 
                 37.449 
                 38.449 
               
               
                 692.862 
                 27.718 
                 28.718 
               
               
                 1245.308 
                 37.446 
                 38.446 
               
               
                 713.5975 
                 24.835 
                 25.835 
               
               
                 766.8335 
                 20.973 
                 21.973 
               
               
                 1118.573 
                 18.91 
                 19.91 
               
               
                 1356.332 
                 40.142 
                 41.142 
               
               
                 713.2632 
                 24.862 
                 25.862 
               
               
                 632.8939 
                 37.449 
                 38.449 
               
               
                 767.3351 
                 20.973 
                 21.973 
               
               
                 1245.354 
                 45.921 
                 46.921 
               
               
                 1092.202 
                 37.164 
                 38.164 
               
               
                 1091.703 
                 37.446 
                 38.446 
               
               
                 576.0089 
                 45.797 
                 46.797 
               
               
                 774.3157 
                 20.963 
                 21.963 
               
               
                 1398.409 
                 37.446 
                 38.446 
               
               
                 1082.377 
                 29.745 
                 30.745 
               
               
                 1082.521 
                 29.72 
                 30.72 
               
               
                 747.7883 
                 28.871 
                 29.871 
               
               
                 747.5877 
                 28.871 
                 29.871 
               
               
                 1017.626 
                 40.143 
                 41.143 
               
               
                 856.5498 
                 27.091 
                 28.091 
               
               
                 1082.234 
                 29.745 
                 30.745 
               
               
                 923.815 
                 27.718 
                 28.718 
               
               
                 514.3178 
                 45.805 
                 46.805 
               
               
                 670.3671 
                 22.036 
                 23.036 
               
               
                 1185.613 
                 29.438 
                 30.438 
               
               
                 534.9825 
                 45.819 
                 46.819 
               
               
                 520.341 
                 45.691 
                 46.691 
               
               
                 747.9889 
                 28.871 
                 29.871 
               
               
                 886.6 
                 30.939 
                 31.939 
               
               
                 1262.604 
                 29.769 
                 30.769 
               
               
                 723.3659 
                 32.732 
                 33.732 
               
               
                 994.2356 
                 45.096 
                 46.096 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Global Parent Masses 65% fraction 
               
               
                 for targeted identification 
               
            
           
           
               
               
               
            
               
                   
                 Start time 
                 End time 
               
               
                 m/z 
                 (min) 
                 (min) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1222.77185 
                 18.898 
                 19.498 
               
               
                 1222.62903 
                 18.898 
                 19.498 
               
               
                 1222.91467 
                 18.898 
                 19.498 
               
               
                 1222.48633 
                 18.898 
                 19.498 
               
               
                 1222.34363 
                 18.898 
                 19.498 
               
               
                 535.41309 
                 44.458 
                 45.058 
               
               
                 549.31537 
                 35.307 
                 35.907 
               
               
                 1240.9231 
                 18.895 
                 19.495 
               
               
                 1241.21008 
                 18.895 
                 19.495 
               
               
                 522.59802 
                 47.752 
                 48.352 
               
               
                 500.20343 
                 24.938 
                 25.538 
               
               
                 557.44525 
                 34.845 
                 35.445 
               
               
                 700.55261 
                 44.458 
                 45.058 
               
               
                 502.29593 
                 31.133 
                 31.733 
               
               
                 576.00928 
                 20.109 
                 20.709 
               
               
                 1229.77344 
                 19.099 
                 19.699 
               
               
                 1227.05896 
                 21.087 
                 21.687 
               
               
                 666.32935 
                 12.86 
                 13.46 
               
               
                 555.42859 
                 44.458 
                 45.058 
               
               
                 919.62494 
                 10.837 
                 11.437 
               
               
                 1086.43494 
                 18.895 
                 19.495 
               
               
                 500.20352 
                 24.16 
                 24.76 
               
               
                 785.54749 
                 44.458 
                 45.058 
               
               
                 1240.49377 
                 18.893 
                 19.493 
               
               
                 656.32324 
                 35.678 
                 36.278 
               
               
                 576.00928 
                 20.962 
                 21.562 
               
               
                 1044.64368 
                 33.755 
                 34.355 
               
               
                 565.43127 
                 34.845 
                 35.445 
               
               
                 534.98254 
                 20.109 
                 20.709 
               
               
                 689.45453 
                 33.647 
                 34.247 
               
               
                 522.59821 
                 46.986 
                 47.586 
               
               
                 552.97772 
                 35.36 
                 35.96 
               
               
                 1160.28918 
                 18.176 
                 18.776 
               
               
                 535.41296 
                 40.034 
                 40.634 
               
               
                 514.31842 
                 22.557 
                 23.157 
               
               
                 1092.1864 
                 19.016 
                 19.616 
               
               
                 1226.62988 
                 21.087 
                 21.687 
               
               
                 1245.21155 
                 21.073 
                 21.673 
               
               
                 538.27802 
                 31.183 
                 31.783 
               
               
                 595.95276 
                 20.109 
                 20.709 
               
               
                 770.53705 
                 35.665 
                 36.265 
               
               
                 514.13129 
                 22.572 
                 23.172 
               
               
                 533.19391 
                 45.359 
                 45.959 
               
               
                 503.29941 
                 31.133 
                 31.733 
               
               
                 1035.65649 
                 33.8 
                 34.4 
               
               
                 1228.77197 
                 19.099 
                 19.699 
               
               
                 865.69196 
                 44.492 
                 45.092 
               
               
                 552.64246 
                 35.36 
                 35.96 
               
               
                 621.2735 
                 35.307 
                 35.907 
               
               
                 639.38116 
                 12.36 
                 12.96 
               
               
                 795.98547 
                 12.411 
                 13.011 
               
               
                 788.02655 
                 34.697 
                 35.297 
               
               
                 816.57715 
                 46.757 
                 47.357 
               
               
                 1245.06909 
                 21.073 
                 21.673 
               
               
                 590.78833 
                 35.36 
                 35.96 
               
               
                 522.59857 
                 46.026 
                 46.626 
               
               
                 1089.55884 
                 16.803 
                 17.403 
               
               
                 785.59174 
                 41.855 
                 42.455 
               
               
                 656.03418 
                 44.963 
                 45.563 
               
               
                 1245.64099 
                 21.073 
                 21.673 
               
               
                 734.5838 
                 41.312 
                 41.912 
               
               
                 527.42432 
                 44.458 
                 45.058 
               
               
                 816.57703 
                 45.912 
                 46.512 
               
               
                 564.90961 
                 44.767 
                 45.367 
               
               
                 1160.14612 
                 18.176 
                 18.776 
               
               
                 787.98962 
                 33.811 
                 34.411 
               
               
                 1530.9856 
                 33.8 
                 34.4 
               
               
                 834.60272 
                 45.536 
                 46.136 
               
               
                 1013.6778 
                 47.807 
                 48.407 
               
               
                 927.50275 
                 24.16 
                 24.76 
               
               
                 770.53809 
                 41.117 
                 41.717 
               
               
                 672.8623 
                 20.478 
                 21.078 
               
               
                 1236.03796 
                 18.898 
                 19.498 
               
               
                 827.44568 
                 17.482 
                 18.082 
               
               
                 1021.62933 
                 31.226 
                 31.826 
               
               
                 612.2973 
                 35.687 
                 36.287 
               
               
                 818.59338 
                 40.929 
                 41.529 
               
               
                 763.073 
                 44.933 
                 45.533 
               
               
                 884.26294 
                 15.568 
                 16.168 
               
               
                 784.58783 
                 34.201 
                 34.801 
               
               
                 647.50586 
                 43.805 
                 44.405 
               
               
                 816.57739 
                 42.456 
                 43.056 
               
               
                 816.57806 
                 44.856 
                 45.456 
               
               
                 589.98645 
                 20.109 
                 20.709 
               
               
                 678.38123 
                 29.773 
                 30.373 
               
               
                 574.37909 
                 36.07 
                 36.67 
               
               
                 590.789 
                 33.644 
                 34.244 
               
               
                 550.38953 
                 39.608 
                 40.208 
               
               
                 1234.76331 
                 21.088 
                 21.688 
               
               
                 747.63464 
                 45.81 
                 46.41 
               
               
                 684.06628 
                 43.942 
                 44.542 
               
               
                 834.60327 
                 43.684 
                 44.284 
               
               
                 1226.48657 
                 21.087 
                 21.687 
               
               
                 537.77429 
                 31.183 
                 31.783 
               
               
                 726.76282 
                 35.307 
                 35.907 
               
               
                 575.44519 
                 44.421 
                 45.021 
               
               
                 856.57281 
                 44.856 
                 45.456 
               
               
                 818.56958 
                 41.989 
                 42.589 
               
               
                 818.59167 
                 37.061 
                 37.661 
               
               
                 780.55658 
                 44.856 
                 45.456 
               
               
                 783.59045 
                 45.191 
                 45.791 
               
               
                 806.57233 
                 36.618 
                 37.218 
               
               
                 547.08124 
                 12.898 
                 13.498 
               
               
                 1255.62939 
                 19.003 
                 19.603 
               
               
                 1101.73071 
                 47.659 
                 48.259 
               
               
                 616.12958 
                 24.863 
                 25.463 
               
               
                 942.46729 
                 24.16 
                 24.76 
               
               
                 1065.6875 
                 33.644 
                 34.244 
               
               
                 564.9295 
                 35.766 
                 36.366 
               
               
                 1096.42273 
                 16.828 
                 17.428 
               
               
                 816.57843 
                 43.658 
                 44.258 
               
               
                 747.63562 
                 42.131 
                 42.731 
               
               
                 606.30951 
                 33.644 
                 34.244 
               
               
                 809.47382 
                 43.611 
                 44.211 
               
               
                 1255.79785 
                 12.391 
                 12.991 
               
               
                 868.50171 
                 39.152 
                 39.752 
               
               
                 1234.90649 
                 21.088 
                 21.688 
               
               
                 789.95789 
                 31.226 
                 31.826 
               
               
                 576.27594 
                 35.36 
                 35.96 
               
               
                 799.41437 
                 15.568 
                 16.168 
               
               
                 528.29279 
                 35.166 
                 35.766 
               
               
                 842.56836 
                 45.191 
                 45.791 
               
               
                 1081.91406 
                 18.898 
                 19.498 
               
               
                 1865.21143 
                 12.45 
                 13.05 
               
               
                 536.73425 
                 10.897 
                 11.497 
               
               
                 800.58289 
                 44.856 
                 45.456 
               
               
                 1761.11316 
                 33.8 
                 34.4 
               
               
                 1234.33362 
                 21.088 
                 21.688 
               
               
                 523.28363 
                 46.596 
                 47.196 
               
               
                 692.56415 
                 44.492 
                 45.092 
               
               
                 856.57227 
                 44.038 
                 44.638 
               
               
                 682.36548 
                 42.931 
                 43.531 
               
               
                 584.9256 
                 45.702 
                 46.302 
               
               
                 508.58325 
                 47.575 
                 48.175 
               
               
                 549.30127 
                 31.216 
                 31.816 
               
               
                 547.81464 
                 35.36 
                 35.96 
               
               
                 640.4176 
                 34.467 
                 35.067 
               
               
                 874.50842 
                 12.645 
                 13.245 
               
               
                 1089.43811 
                 21.069 
                 21.669 
               
               
                 834.58734 
                 44.106 
                 44.706 
               
               
                 548.95966 
                 20.109 
                 20.709 
               
               
                 811.67133 
                 44.9 
                 45.5 
               
               
                 977.78485 
                 43.805 
                 44.405 
               
               
                 984.71124 
                 45.034 
                 45.634 
               
               
                 816.57745 
                 39.918 
                 40.518 
               
               
                 541.35706 
                 37.363 
                 37.963 
               
               
                 1242.32043 
                 21.087 
                 21.687 
               
               
                 1296.89185 
                 18.895 
                 19.495 
               
               
                 816.57672 
                 41.217 
                 41.817 
               
               
                 834.60321 
                 42.206 
                 42.806 
               
               
                 800.58289 
                 36.618 
                 37.218 
               
               
                 1057.11133 
                 31.226 
                 31.826 
               
               
                 841.43475 
                 46.467 
                 47.067 
               
               
                 1090.30103 
                 18.898 
                 19.498 
               
               
                 1076.55383 
                 19.11 
                 19.71 
               
               
                 516.23901 
                 44.751 
                 45.351 
               
               
                 699.44244 
                 34.996 
                 35.596 
               
               
                 1082.91907 
                 19.11 
                 19.71 
               
               
                 816.57849 
                 36.279 
                 36.879 
               
               
                 1073.30225 
                 21.087 
                 21.687 
               
               
                 836.44843 
                 35.316 
                 35.916 
               
               
                 928.77789 
                 43.805 
                 44.405 
               
               
                 500.30814 
                 33.647 
                 34.247 
               
               
                 1096.2981 
                 16.79 
                 17.39 
               
               
                 1252.44897 
                 19.099 
                 19.699 
               
               
                 800.5827 
                 37.369 
                 37.969 
               
               
                 797.4433 
                 31.183 
                 31.783 
               
               
                 780.55627 
                 41.566 
                 42.166 
               
               
                 997.70264 
                 47.786 
                 48.386 
               
               
                 1207.7627 
                 18.983 
                 19.583 
               
               
                 847.11377 
                 44.569 
                 45.169 
               
               
                 1512.69934 
                 18.898 
                 19.498 
               
               
                 1856.21155 
                 12.469 
                 13.069 
               
               
                 1250.02783 
                 19.099 
                 19.699 
               
               
                 1095.60803 
                 33.811 
                 34.411 
               
               
                 658.4317 
                 36.611 
                 37.211 
               
               
                 1098.92664 
                 19.016 
                 19.616 
               
               
                 972.04376 
                 11.007 
                 11.607 
               
               
                 571.61591 
                 31.37 
                 31.97 
               
               
                 561.2981 
                 31.327 
                 31.927 
               
               
                 591.93182 
                 39.863 
                 40.463 
               
               
                 800.58289 
                 39.551 
                 40.151 
               
               
                 1309.29358 
                 31.226 
                 31.826 
               
               
                 817.58173 
                 41.855 
                 42.455 
               
               
                 650.42218 
                 31.629 
                 32.229 
               
               
                 591.38416 
                 35.266 
                 35.866 
               
               
                 550.34637 
                 36.076 
                 36.676 
               
               
                 507.32535 
                 32.394 
                 32.994 
               
               
                 1242.32202 
                 19.128 
                 19.728 
               
               
                 1452.40747 
                 16.828 
                 17.428 
               
               
                 640.44788 
                 36.711 
                 37.311 
               
               
                 1296.60388 
                 18.899 
                 19.499 
               
               
                 574.38922 
                 39.095 
                 39.695 
               
               
                 1127.66003 
                 35.36 
                 35.96 
               
               
                 549.04468 
                 10.94 
                 11.54 
               
               
                 1288.52576 
                 20.914 
                 21.514 
               
               
                 1452.41113 
                 21.087 
                 21.687 
               
               
                 943.24799 
                 33.642 
                 34.242 
               
               
                 1244.78503 
                 21.069 
                 21.669 
               
               
                 1236.81531 
                 12.778 
                 13.378 
               
               
                 656.0343 
                 43.815 
                 44.415 
               
               
                 552.31799 
                 33.644 
                 34.244 
               
               
                 533.19354 
                 44.604 
                 45.204 
               
               
                 800.58374 
                 38.715 
                 39.315 
               
               
                 800.58313 
                 41.099 
                 41.699 
               
               
                 1105.16418 
                 19.016 
                 19.616 
               
               
                 1080.5448 
                 19.042 
                 19.642 
               
               
                 1234.19116 
                 21.088 
                 21.688 
               
               
                 834.58575 
                 37.992 
                 38.592 
               
               
                 722.05969 
                 44.8 
                 45.4 
               
               
                 1537.02759 
                 33.8 
                 34.4 
               
               
                 542.90161 
                 44.569 
                 45.169 
               
               
                 1441.04272 
                 18.895 
                 19.495 
               
               
                 1057.70325 
                 34.656 
                 35.256 
               
               
                 575.38568 
                 44.131 
                 44.731 
               
               
                 528.40558 
                 36.809 
                 37.409 
               
               
                 694.05194 
                 43.783 
                 44.383 
               
               
                 591.98376 
                 21.656 
                 22.256 
               
               
                 780.55603 
                 42.334 
                 42.934 
               
               
                 832.57202 
                 40.929 
                 41.529 
               
               
                 708.03638 
                 44.492 
                 45.092 
               
               
                 743.07135 
                 41.312 
                 41.912 
               
               
                 731.60846 
                 42.622 
                 43.222 
               
               
                 1350.76477 
                 38.534 
                 39.134 
               
               
                 548.95728 
                 33.799 
                 34.399 
               
               
                 816.57764 
                 35.123 
                 35.723 
               
               
                 1080.66956 
                 21.088 
                 21.688 
               
               
                 1063.85815 
                 20.109 
                 20.709 
               
               
                 742.09894 
                 35.339 
                 35.939 
               
               
                 527.31049 
                 33.782 
                 34.382 
               
               
                 585.40204 
                 33.044 
                 33.644 
               
               
                 859.44659 
                 35.307 
                 35.907 
               
               
                 1080.41858 
                 21.09 
                 21.69 
               
               
                 818.59222 
                 34.562 
                 35.162 
               
               
                 1370.99316 
                 44.806 
                 45.406 
               
               
                 1089.53223 
                 19.11 
                 19.71 
               
               
                 1431.85144 
                 12.411 
                 13.011 
               
               
                 695.89008 
                 20.593 
                 21.193 
               
               
                 591.42761 
                 41.789 
                 42.389 
               
               
                 504.75061 
                 31.022 
                 31.622 
               
               
                 968.62842 
                 39.552 
                 40.152 
               
               
                 863.56744 
                 43.589 
                 44.189 
               
               
                 1439.88672 
                 21.088 
                 21.688 
               
               
                 809.54089 
                 40.947 
                 41.547 
               
               
                 1234.05066 
                 21.049 
                 21.649 
               
               
                 1080.41943 
                 19.099 
                 19.699 
               
               
                 1259.47473 
                 20.829 
                 21.429 
               
               
                 1251.28943 
                 12.43 
                 13.03 
               
               
                 1874.19434 
                 12.428 
                 13.028 
               
               
                 1098.1825 
                 12.403 
                 13.003 
               
               
                 678.40588 
                 35.36 
                 35.96 
               
               
                 1080.2937 
                 21.09 
                 21.69 
               
               
                 1163.60168 
                 31.331 
                 31.931 
               
               
                 1081.90649 
                 21.003 
                 21.603 
               
               
                 1303.35498 
                 20.914 
                 21.514 
               
               
                 730.01355 
                 37.502 
                 38.102 
               
               
                 540.86346 
                 41.855 
                 42.455 
               
               
                 627.93677 
                 39.175 
                 39.775 
               
               
                 1226.34363 
                 21.087 
                 21.687 
               
               
                 754.50586 
                 44.569 
                 45.169 
               
               
                 820.47766 
                 35.368 
                 35.968 
               
               
                 1440.05261 
                 21.087 
                 21.687 
               
               
                 763.05652 
                 39.17 
                 39.77 
               
               
                 965.57751 
                 35.3 
                 35.9 
               
               
                 956.92969 
                 18.895 
                 19.495 
               
               
                 549.7619 
                 33.862 
                 34.462 
               
               
                 1039.28918 
                 32.404 
                 33.004 
               
               
                 1027.18225 
                 38.565 
                 39.165 
               
               
                 540.86285 
                 40.956 
                 41.556 
               
               
                 1220.05237 
                 18.899 
                 19.499 
               
               
                 646.42871 
                 33.65 
                 34.25 
               
               
                 1864.20129 
                 12.391 
                 12.991 
               
               
                 1279.36121 
                 18.902 
                 19.502 
               
               
                 1501.39685 
                 18.898 
                 19.498 
               
               
                 1238.34937 
                 20.516 
                 21.116 
               
               
                 1252.34387 
                 20.983 
                 21.583 
               
               
                 1425.90979 
                 33.836 
                 34.436 
               
               
                 1087.41003 
                 19.128 
                 19.728 
               
               
                 1356.00232 
                 16.785 
                 17.385 
               
               
                 804.55017 
                 40.49 
                 41.09 
               
               
                 1611.92188 
                 31.276 
                 31.876 
               
               
                 650.42383 
                 33.647 
                 34.247 
               
               
                 1238.32214 
                 17.718 
                 18.318 
               
               
                 795.48767 
                 35.162 
                 35.762 
               
               
                 868.92645 
                 31.353 
                 31.953 
               
               
                 1664.72192 
                 12.411 
                 13.011 
               
               
                 1260.61768 
                 21.069 
                 21.669 
               
               
                 1159.58667 
                 46.467 
                 47.067 
               
               
                 741.53467 
                 37.131 
                 37.731 
               
               
                 1266.21619 
                 18.902 
                 19.502 
               
               
                 1275.7948 
                 33.733 
                 34.333 
               
               
                 1245.63 
                 20.983 
                 21.583 
               
               
                 696.51019 
                 44.963 
                 45.563 
               
               
                 1089.3103 
                 21.087 
                 21.687 
               
               
                 704.9386 
                 43.649 
                 44.249 
               
               
                 1178.38953 
                 35.3 
                 35.9 
               
               
                 811.95068 
                 10.634 
                 11.234 
               
               
                 751.05286 
                 44.8 
                 45.4 
               
               
                 936.49298 
                 31.271 
                 31.871 
               
               
                 737.05133 
                 44.458 
                 45.058 
               
               
                 939.39587 
                 24.473 
                 25.073 
               
               
                 1027.66821 
                 33.8 
                 34.4 
               
               
                 714.42725 
                 39.557 
                 40.157 
               
               
                 780.98224 
                 35.166 
                 35.766 
               
               
                 834.58661 
                 41.639 
                 42.239 
               
               
                 571.37 
                 39.418 
                 40.018 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                   
                 Start 
                 End 
               
               
                 MZ 
                 Time 
                 Time 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 502.2947 
                 30.953 
                 31.953 
               
               
                 576.0092 
                 17.85 
                 18.85 
               
               
                 1035.655 
                 33.622 
                 34.622 
               
               
                 1021.629 
                 31.026 
                 32.026 
               
               
                 787.9893 
                 33.601 
                 34.601 
               
               
                 534.9822 
                 17.85 
                 18.85 
               
               
                 1530.986 
                 33.601 
                 34.601 
               
               
                 666.3301 
                 12.673 
                 13.673 
               
               
                 789.9586 
                 31.016 
                 32.016 
               
               
                 1027.67 
                 33.601 
                 34.601 
               
               
                 1309.292 
                 31.026 
                 32.026 
               
               
                 595.9525 
                 17.85 
                 18.85 
               
               
                 780.982 
                 35.033 
                 36.033 
               
               
                   
               
            
           
         
       
     
     Proteome Discoverer 1.1 was used to identify the differentially expressed peptides with the work flow as follows: 
     
       
         
           
               
             
               
                 TABLE 9 
               
               
                   
               
               
                 Input Data 
               
               
                   
               
             
            
               
                 1. General Settings 
               
            
           
           
               
               
               
            
               
                   
                 Precursor Selection 
                 Use MS1 Precursor 
               
            
           
           
               
            
               
                 2. Spectrum Properties Filter 
               
            
           
           
               
               
               
            
               
                   
                 Lower RT Limit 
                 5 
               
               
                   
                 Upper RT Limit 
                 84  
               
               
                   
                 Lowest Charge State 
                 1 
               
               
                   
                 Highest Charge State 
                 4 
               
               
                   
                 Min. Precursor Mass 
                  100 Da 
               
               
                   
                 Max. Precursor Mass 
                 9000 Da 
               
               
                   
                 Total Intensity Threshold 
                 0 
               
               
                   
                 Minimum Peak Count 
                 1 
               
            
           
           
               
            
               
                 3. Scan Event Filters 
               
            
           
           
               
               
               
            
               
                   
                 Mass Analyzer 
                 Is ITMS; FTMS 
               
               
                   
                 MS Order 
                 Is MS2 
               
               
                   
                 Activation Type 
                 Is CID 
               
               
                   
                 Scan Type 
                 Is Full 
               
               
                   
                 Ionization Source 
                 Is ESI 
               
               
                   
                 Polarity Mode 
                 Is+ 
               
            
           
           
               
            
               
                 3. Peak Filters 
               
            
           
           
               
               
               
            
               
                   
                 S/N Threshold 
                 0 
               
            
           
           
               
            
               
                 4. Replacement for Unrecognized Properties 
               
            
           
           
               
               
               
            
               
                   
                 Unrecognized Charge Re 
                 1; 2; 3; 4 
               
               
                   
                 Unrecognized Mass Anal 
                 ITMS 
               
               
                   
                 Unrecognized MS Order 
                 MS2 
               
               
                   
                 Unrecognized Activation 
                 CID 
               
               
                   
                 Unrecognized Polarity 
                 + 
               
            
           
           
               
            
               
                 1. Spectrum Match Criteria 
               
            
           
           
               
               
               
            
               
                   
                 Precursor Mass Criterion 
                 Same Measured M 
               
               
                   
                 Presursor Mass Tolerance 
                 7 ppm 
               
               
                   
                 Max. RT Difference [min] 
                   1.5 
               
               
                   
                 Allow Mass Analyzer Mis 
                 False 
               
               
                   
                 Allow MS Order Mismatch 
                 False 
               
            
           
           
               
            
               
                 1. Thresholds 
               
            
           
           
               
               
               
            
               
                   
                 S/N Threshold 
                 0 
               
            
           
           
               
            
               
                 1. Filter Settings 
               
            
           
           
               
               
               
            
               
                   
                 Mass Analyzer 
                 Is ITMS; FTMS 
               
               
                   
                 MS Order 
                 Is MS1; MS2 
               
               
                   
                 Activation Type 
                 Is CID 
               
               
                   
                 Scan Type 
                 Is Full 
               
               
                   
                 Ionization Source 
                 Is ESI 
               
               
                   
                 Polarity Mode 
                 Is+ 
               
            
           
           
               
            
               
                 1. Spectrum Properties 
               
            
           
           
               
               
               
            
               
                   
                 Lowest Charge State 
                 1 
               
               
                   
                 Highest Charge State 
                 4 
               
               
                   
                 Min. Precursor Mass 
                  100 Da 
               
               
                   
                 Max. Precursor Mass 
                 9000 Da 
               
            
           
           
               
            
               
                 2. Thresholds 
               
            
           
           
               
               
               
            
               
                   
                 Total Intensity Threshold 
                 0 
               
               
                   
                 Minimum Peak Count 
                 1 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
               
                   
               
             
            
               
                 1. Input Data 
               
            
           
           
               
               
               
            
               
                   
                 Protein Database 
                 Maha.fasta 
               
               
                   
                 Enzyme Name 
                 No-Enzyme [No 
               
               
                   
                 Maximum Missed Cleavage 
                 0 
               
            
           
           
               
            
               
                 2. Decoy Database Search 
               
            
           
           
               
               
               
            
               
                   
                 Search Against Decoy D 
                 False 
               
               
                   
                 Target FDR (Strict) 
                 0.01 
               
               
                   
                 Target FDR (Relaxed) 
                 0.05 
               
            
           
           
               
            
               
                 3. Tolerances 
               
            
           
           
               
               
               
            
               
                   
                 Precursor Mass Tolerance 
                   7 ppm 
               
               
                   
                 Fragment Mass Tolerance 
                 0.8 Da 
               
               
                   
                 Use Average Precursor 
                 False 
               
               
                   
                 Use Average Fragment 
                 False 
               
            
           
           
               
            
               
                 4. Ion Series 
               
            
           
           
               
               
               
            
               
                   
                 Use Neutral Loss a Ions 
                 True 
               
               
                   
                 Use Neutral Loss b Ions 
                 True 
               
               
                   
                 Use Neutral Loss y Ions 
                 True 
               
               
                   
                 Weight of a Ions 
                 0 
               
               
                   
                 Weight of b Ions 
                 1 
               
               
                   
                 Weight of c Ions 
                 0 
               
               
                   
                 Weight of x Ions 
                 0 
               
               
                   
                 Weight of y Ions 
                 1 
               
               
                   
                 Weight of z Ions 
                 0 
               
            
           
           
               
            
               
                 5. Dynamic Modifications 
               
            
           
           
               
               
               
            
               
                   
                 N-Terminal Modification 
                 None 
               
               
                   
                 C-Terminal Modification 
                 None 
               
               
                   
                 1. Dynamic Modification 
                 None 
               
               
                   
                 2. Dynamic Modification 
                 None 
               
               
                   
                 3. Dynamic Modification 
                 None 
               
               
                   
                 4. Dynamic Modification 
                 None 
               
               
                   
                 5. Dynamic Modification 
                 None 
               
               
                   
                 6. Dynamic Modification 
                 None 
               
            
           
           
               
            
               
                 6. Static Modifications 
               
            
           
           
               
               
               
            
               
                   
                 Peptide N-Terminus 
                 None 
               
               
                   
                 Peptide C-Terminus 
                 None 
               
               
                   
                   
               
            
           
         
       
     
     The database for peptide annotation was created from NCBI, Swissprot, and Uniprot. The resulting annotated proteins are provided above in Table 1. 
     Example 3 
     Inosine Concentrations in Dogs with Renal Disease 
     Dog serum was obtained from field samples submitted to IDEXX Reference Laboratories. Dogs were of various breeds and ages. 25 samples with &lt;1.8 mg/dL serum creatinine were assigned to a low creatinine group, and 25 samples with &gt;1.8 mg/dL serum creatinine were assigned to a high creatinine group. Again, high creatinine is associated with renal disease, therefore inosine levels were assessed to determine whether inosine could be a biomarker for reduced kidney function. 
     Serum samples from a high creatinine and normal creatinine canine populations were analyzed on LC/MS and differentially produced mass features were identified by informatics as previously described. LC/MS was run for each sample (i.e., dog) individually. SIEVE software (Thermo Scientific, Waltham, Mass.) was used for statistical analysis of the LC/MS data. Raw LC/MS data files were loaded into SIEVE, and peaks were identified. Statistical analysis was performed to compare peaks in low creatinine and high creatinine samples. A differential peak corresponding to inosine was identified. Serum inosine was found to be depleted in 13 out of the 25 dogs with high serum creatinine. The ion intensity for inosine (as measured by LC/MS) is shown in  FIG.  1   , where “Renal” represents the 13 dogs with high creatinine and inosine depletion, and “Control” represents all 25 dogs with low serum creatinine. 
     A protocol utilized for initial LC/MS analysis as shown in  FIG.  1    follows below: Plasma extraction was performed in a 0.5 mL protein LoBind eppendorf tube. 110 uL of canine serum was precipitated by addition of 200 uL acetonitrile. After vortexing for 10 seconds, and leaving the sample at room temperature for 30 minutes, the precipitate was pelleted by centrifugation at 13,000 rpm for 30 minutes at room temperature using a benchtop centrifuge. The supernatant was then analyzed by LC/MS. SIEVE and R were used to identify molecules present at differential levels (p-value &lt;0.05). 
     LC method was performed with Solvent A: 0.1% Formic acid in water and Solvent B: 0.1% Formic acid in acetonitrile: 
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                   
                   
               
               
                   
                 No 
                 Time 
                 A % 
                 B % 
                 C % 
                 D % 
                 μL/min 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 1 
                 0 
                 100 
                 0 
                 0 
                 0 
                 300 
               
               
                   
                 2 
                 5 
                 100 
                 0 
                 0 
                 0 
                 300 
               
               
                   
                 3 
                 23 
                 65 
                 35 
                 0 
                 0 
                 300 
               
               
                   
                 4 
                 26 
                 65 
                 35 
                 0 
                 0 
                 300 
               
               
                   
                 5 
                 44 
                 5 
                 95 
                 0 
                 0 
                 300 
               
               
                   
                 6 
                 46 
                 5 
                 95 
                 0 
                 0 
                 300 
               
               
                   
                 7 
                 46.5 
                 100 
                 0 
                 0 
                 0 
                 300 
               
               
                   
                 8 
                 60 
                 100 
                 0 
                 0 
                 0 
                 300 
               
               
                   
                   
               
            
           
         
       
         
         Column: Acquity UPLC BEH130 C18 1.7 μM 2.1id×150 mm length 
         Guard Column: vanguard BEH 300 C 18  1.7 uM 
         Injection volume: 25 μL 
         Tray temp: 10° C. 
         Column oven temp: 45° C. 
         MS run time: 60 minutes 
         Divert valve:
       To waste 0-5   To source 5-55   To waste 55-60   
     
       
    
     Mass Spectrometry method was performed according to the following parameters:
     MS scan event 1: FTMS; resolution 30000; scan range 100.0-500.0   MS scan event 2: FTMS; resolution 30000; scan range 500.0-2000.0   MS Tune File Values   Source Type: ESI   Capillary Temp (° C.): 250.00   Sheath gas Flow: 24.00   Aux Gas Flow: 13.00   Sweep Gas Flow: 0   Ion Trap MSn AGC Target: 10000   FTMS Injection waveforms: off   FTMS AGC Target: 500000   Source voltage (kV): 4.50   Source current (μA): 100.00   Capillary Voltage (V): 68.28   Tube Lens (V): 130.00   Skimmer Offset (V): 0.00   Multipole RF Amplifier (Vp-p): 550.00   Multipole 00 offset (V): −1.60   Lens 0 Voltage (V): −2.70   Multipole 0 offset (V): −5.80   Lens 1 Voltage (V): −11.00   Gate Lens offset (V): −60.00   Multipole 1 offset (V): −10.50   Front Lens (V): −5.18   FTMS full microscans: 1   FTMS full Max Ion Time (ms): 500   Ion Trap MSn Micro Scans: 3   Ion Trap MSn Max Ion Time: 100   

     To verify inosine as a biomarker for kidney disease, a complementary study was performed on dogs with X-linked hereditary nephropathy (XLHN). XLHN is caused by a mutation in the gene COL4A5 (see Example 1 for details). These XLHN dogs provided a model of kidney disease that begins as glomerular defect and progresses to tubular failure. Serum and urine samples from four male dog puppies with XLHN (Table 11) were collected at pre-disease, mid-stage, and end-stage disease and analyzed for inosine as described in the Renal LC/MS Assay provided below. 
     LC/MS Mobile Phases Prep. 
     
         
         
           
             1. Mobile Phase A: to 1 liter of water add 1 ml acetic acid. Mix well. 
             2. Mobile Phase B: to 1 liter of Acetonitrile add 1 ml of acetic acid. Mix well. 
           
         
       
    
     Internal STD (IS Solution) Prep 
     
         
         
           
             1. Weigh 5 mg deuterated creatinine and 6-Chloropurine riboside into a 20 ml vial. 
             2. Add 5 ml of water to dilute. (1 mg/ml solution). 
             3. Transfer 5 ml of #2 into a 21 flask and add 21 of water to the mark (2.5 ug/ml solution). 
             4. Use #3 as internal STD spiking solution. 
           
         
       
    
     STD Curve Prep 
     
         
         
           
             1. Weigh 10 mg creatinine and 10 mg inosine into a 2 ml vial and add 10 ml of Water to dissolve (1 mg/ml solution). 
             2. Weigh 345 mg of Bovine Serum albumin (BSA) into 5 ml of phosphate buffer saline solution. Mix well. Scale up or down as needed (PBS-BSA Solution). 
             3. Transfer 5 ul of 1 mg/ml solution into 990 ul of PBS-BSA solution (5 ug/ml STD point1) 
             4. Make 11 l/l serial dilutions of #3 for STD points, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and a blank. 
           
         
       
    
     Sample Prep 
     
         
         
           
             1. Thaw serum samples. 
             2. Vortex samples for 10 secs then centrifuge at 3000×g at room temperature for 10 min. 
             3. Transfer 50 ul of samples and STD curve points into microfuge tubes or 96 well plate. 
             4. Add 50 ul of IS solution into each sample. 
             5. Add 100 ul of Acetonitrile. 
             6. Vortex to mix. 
             7. Sonicate for 20 min in water bath. 
             8. Centrifuge at 3000×g for 20 min at 25 degrees c. 
             9. Filter supernatant into amber vials/96 well plates using 0.4 micron nylon filters. 
             10. Analyze samples by LC/MS. 
           
         
       
    
     LC/MS METHOD 
       
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 Column 
                 50 × 4.6 XBridge Amide, 3.5 um column 
               
               
                 Flow 
                 1 ml/min 
               
               
                   
               
            
           
           
               
            
               
                 Gradient 
               
            
           
           
               
               
               
               
               
            
               
                 Step 
                 total time 
                 flow rate (ul/ml) 
                 A % 
                 B % 
               
               
                   
               
               
                 0. 
                 0.1 
                 1000 
                 20 
                 80 
               
               
                 1. 
                 5.0 
                 1000 
                 100 
                 0 
               
               
                 2. 
                 8.00 
                 1000 
                 100 
                 0 
               
               
                 3. 
                 8.10 
                 1000 
                 20 
                 80 
               
               
                 4. 
                 14.00 
                 1000 
                 20 
                 80 
               
               
                   
               
            
           
           
               
               
            
               
                 Time 
                 14 min 
               
               
                 Temperature 
                 ambient 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                   
               
               
                 MS Parameters 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Scan Type: 
                 MRM 
               
               
                   
                 Polarity: 
                 Positive 
               
               
                   
                 Scan Mode: 
                 N/A 
               
               
                   
                 Ion Source: 
                 Turbo Spray 
               
               
                   
                 Resolution Q1: 
                 Unit 
               
               
                   
                 Resolution Q3: 
                 Unit 
               
            
           
           
               
               
               
               
            
               
                   
                 Intensity Thres.: 
                 0.00  
                 cps 
               
               
                   
                 Settling time: 
                 0.000  
                 msec 
               
               
                   
                 MR pause: 
                 5.000  
                 msec 
               
            
           
           
               
               
               
            
               
                   
                 MCA: 
                 No 
               
            
           
           
               
               
               
               
            
               
                   
                 Step size: 
                 0.00  
                 amu 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                 Q1 Mass (amu) 
                 Q3 Mass (amu) 
                 Dwell (msec) 
                 Parameters 
                 Value 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 Inosine 
               
            
           
           
               
               
               
               
               
            
               
                 269.1 
                 137.1 
                 150.00 
                 DP 
                 30 
               
               
                   
                   
                   
                 EP 
                 7 
               
               
                   
                   
                   
                 CEP 
                 8 
               
               
                   
                   
                   
                 CE 
                 17 
               
               
                   
                   
                   
                 CXP 
                 3 
               
            
           
           
               
            
               
                 CREATININE 
               
            
           
           
               
               
               
               
               
            
               
                 114.20 
                 44.2 
                 150.00 
                 DP 
                 20 
               
               
                   
                   
                   
                 EP 
                 6.30 
               
               
                   
                   
                   
                 CEP 
                 8.34 
               
               
                   
                   
                   
                 CE 
                 35 
               
               
                   
                   
                   
                 CXP 
                 4 
               
            
           
           
               
            
               
                 DEUTERATED CREATININE 
               
            
           
           
               
               
               
               
               
            
               
                 117.20 
                 47.2 
                 150.00 
                 DP 
                 20 
               
               
                   
                   
                   
                 EP 
                 6.30 
               
               
                   
                   
                   
                 CEP 
                 8.47 
               
               
                   
                   
                   
                 CE 
                 35 
               
               
                   
                   
                   
                 CXP 
                 4 
               
            
           
           
               
            
               
                 6-CHLOROPURINE RIBOSIDE 
               
            
           
           
               
               
               
               
               
            
               
                 285.29 
                 153.2 
                 150.00 
                 DP 
                 30 
               
               
                   
                   
                   
                 EP 
                 7 
               
               
                   
                   
                   
                 CEP 
                 8 
               
               
                   
                   
                   
                 CE 
                 17 
               
               
                   
                   
                   
                 CXP 
                 3 
               
               
                   
               
            
           
         
       
     
     The inosine concentrations identified as a result of the above analysis are shown in Table 11, where serum inosine and urine inosine are shown in ug/dL, and creatinine is shown in mg/dL. A significant decrease in inosine is reflected in each animal over time as kidney disease progresses. These data confirm the role of inosine as a biomarker for kidney disease and tubular failure. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Inosine Levels in Dogs with XLHN 
               
            
           
           
               
               
               
               
               
            
               
                 Animal ID 
                 DAY 
                 Serum Inosine 
                 Urine Inosine 
                 Serum Creatinine 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 RASCAL 
                 0 
                 217.03 
                 182.16 
                 0.34 
               
               
                   
                 84 
                 188.54 
                 44.30 
                 1.88 
               
               
                   
                 119 
                 37.10 
                 25.99 
                 3.02 
               
               
                 SANTANA 
                 0 
                 288.08 
                 167.91 
                 0.41 
               
               
                   
                 56 
                 241.82 
                 48.45 
                 1.17 
               
               
                   
                 99 
                 85.80 
                 33.92 
                 6.47 
               
               
                 STEEL 
                 0 
                 174.74 
                 556.90 
                 0.35 
               
               
                   
                 87 
                 128.38 
                 N/D 
                 1.84 
               
               
                   
                 147 
                 11.25 
                 199.25 
                 4.01 
               
               
                 XELLUS 
                 0 
                 115.96 
                 2335.26 
                 0.74 
               
               
                   
                 91 
                 59.87 
                 N/D 
                 1.88 
               
               
                   
                 129 
                 40.61 
                 1640.90 
                 4.05 
               
               
                   
               
            
           
         
       
     
     Example 4 
     Renal Disease Progression in XLHN 
     Patient blood samples were collected from heterozygous female XLHN dogs as described in Example 1. The samples were prepared as described in Example 1 with the exception that all fractions were eluted in 0.1% formic acid in 35% acetonitrile/water, and that the samples were reconstituted in 0.1% formic acid in 3.5% acetonitrile/water. 
     The samples were then subjected to LC/MS as described in Example 2 above, except that the tray temperature was 10 degrees Celsius and the MS run time was 60 minutes. Table 12 shows the results of LC/MS measurements of five peptides (SEQ ID NO:1 (Apolipoprotein C1); SEQ ID NO:31 (Cystatin A); SEQ ID NO:18 (Fibrinogen α chain); SEQ ID NO:25 (Inter-Alpha Inhibitor H4 (ITIH4)); SEQ ID NO:23 (Kininogen) over time, in four heterozygous female XLHN dogs. In Table 12, “NF” is an abbreviation for “not found” (i.e. below the limit of detection), while “ND” is an abbreviation for “not determined”. As the kidney disease progressed, ApoC1 and Inter-Alpha Inhibitor H4 (ITIH4) levels increased, while Fibrinogen alpha levels decreased. Kininogen levels were higher in the XLHN dogs than in the control dogs. Cystatin A levels were higher in at least three out of the four XLHN dogs as compared to the control dogs. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Peptide Levels During Renal Disease Progression 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Inter-Alpha 
                   
                   
               
               
                   
                   
                   
                   
                   
                 Inhibitor 
                   
                   
               
               
                   
                   
                   
                   
                 Fibrinogen α 
                 H4 
                   
                   
               
               
                   
                   
                 Apolipoprotein 
                 Cystatin A 
                 chain 
                 (ITIH4) 
                 Kininogen 
                   
               
               
                   
                   
                 C1 
                 KA-17 
                 EV-11 
                 GV-22 
                 EQ-9 
                 Serum 
               
               
                   
                   
                 AA-26 
                 (SEQ ID 
                 (SEQ ID 
                 (SEQ ID 
                 (SEQ ID 
                 Creatinine 
               
               
                 Animal ID 
                 Age 
                 (SEQ ID NO: 1) 
                 NO: 31) 
                 NO: 18) 
                 NO: 25) 
                 NO: 23) 
                 (mg/dl) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 CONTROL 1 
                 3-4 
                 NF 
                 NF 
                 4386.5 
                 5.9 
                 NF 
                 ND 
               
               
                   
                 months 
                   
                   
                   
                   
                   
                   
               
               
                   
                 Old 
                   
                   
                   
                   
                   
                   
               
               
                 CONTROL 2 
                 3-4 
                 20.6 
                 NF 
                 3881.7 
                 2.2 
                 NF 
                 ND 
               
               
                   
                 months 
                   
                   
                   
                   
                   
                   
               
               
                   
                 Old 
                   
                   
                   
                   
                   
                   
               
               
                 CONTROL 3 
                 3-4 
                 17.7 
                 NF 
                 2344.1 
                 3.6 
                 NF 
                 ND 
               
               
                   
                 months 
                   
                   
                   
                   
                   
                   
               
               
                   
                 Old 
                   
                   
                   
                   
                   
                   
               
               
                 CONTROL 4 
                 3-4 
                 22.3 
                 NF 
                 3741.2 
                 4.3 
                 NF 
                 ND 
               
               
                   
                 months 
                   
                   
                   
                   
                   
                   
               
               
                   
                 Old 
                   
                   
                   
                   
                   
                   
               
               
                 RASCAL 
                 0 
                 114.4 
                 5.2 
                 6712.9 
                 26.2 
                 42.8 
                 0.34 
               
               
                 RASCAL 
                 84 
                 321.6 
                 NF 
                 6819.3 
                 92.3 
                 66.5 
                 1.88 
               
               
                 RASCAL 
                 119 
                 247.1 
                 2.7 
                 3741.2 
                 108.1 
                 19.4 
                 3.02 
               
               
                 XELLUS 
                 0 
                 122.8 
                 NF 
                 4233.3 
                 58.6 
                 10.7 
                 0.74 
               
               
                 XELLUS 
                 91 
                 145.8 
                 NF 
                 3144.7 
                 53.0 
                 1.2 
                 1.88 
               
               
                 XELLUS 
                 129 
                 218.6 
                 NF 
                 2595.7 
                 99.0 
                 16.4 
                 4.05 
               
               
                 SANTANA 
                 0 
                 152.6 
                 9.8 
                 9439.1 
                 62.2 
                 26.7 
                 0.41 
               
               
                 SANTANA 
                 56 
                 149.7 
                 30.9 
                 8811.6 
                 76.6 
                 31.0 
                 1.17 
               
               
                 SANTANA 
                 99 
                 202.4 
                 28.2 
                 7140.7 
                 110.9 
                 17.6 
                 6.46 
               
               
                 STEEL 
                 0 
                 110.9 
                 5.9 
                 12354.8 
                 58.4 
                 21.3 
                 0.35 
               
               
                 STEEL 
                 87 
                 210.9 
                 12.6 
                 8246.6 
                 85.0 
                 38.3 
                 1.84 
               
               
                 STEEL 
                 147 
                 305.3 
                 NF 
                 6628.9 
                 71.4 
                 21.5 
                 4.01 
               
               
                   
               
            
           
         
       
     
     Example 5 
     Renal-Failure Induced Canine Model 
     Dogs of mixed breeds and sizes were injected with dichromate, inducing acute renal failure, specifically due to tubular injury. See Ruegg et al., Toxicol Appl Pharmacol. 1987, 90(2):261-7; Pedraza-Chaverri et al., BMC Nephrology 2005, 6:4; Chiusolo et al., Toxicol Pathol. 2010, 38:338-45. Specifically, dogs were injected with 0.2 mL/kg of potassium dichromate (5 mg/ml). Serum was prepared from blood samples collected at various time points. NGAL (neutrophil gelatinase-associated lipocalin) was assayed with the Dog NGAL ELISA Kit (BioPorto Diagnostics, Gentofte, Denmark) according to the manufacturer&#39;s instructions. Inosine concentrations were measured in serum derived from blood samples taken at various times after injection of dichromate. Inosine and creatinine were measured by LC/MS as previously described in the preceding Example (Renal Assay LC/MS). 
     A time course of inosine, creatinine and NGAL levels following dichromate injection in a single dog is shown in  FIG.  2   . Inosine concentrations dropped within 2 hours of dichromate treatment. Between about 60 and 70 hours post-treatment, inosine levels began to recover. See, Fatima, et al., Hum Exp Toxicol 2005, 24:631-8. Creatinine and NGAL were included as reference markers ( FIG.  2   ). In summary, these data illustrate that reduced inosine levels provide a marker for renal failure and tubular injury. 
     In an additional study, serum samples from dichromate-treated dogs were prepared and subjected to LC/MS as described above in Example 4.  FIG.  3    shows time course measurements of the relative concentrations of three peptides (SEQ ID NO:1 (Apolipoprotein C1); SEQ ID NO:23 (Kininogen); SEQ ID NO:25 (Inter-Alpha Inhibitor H4 (ITIH4))) in two dogs. 
     SEQ ID NO:1 (Apolipoprotein C1) levels increased between about 4 hours and about 48 hours of dichromate treatment ( FIG.  3 A ). Between about 84 and 108 hours post-treatment, peptide SEQ ID NO:1 (Apolipoprotein C1) levels began to recover (decrease). These data illustrate that increased SEQ ID NO:1 (Apolipoprotein C1) levels provide a marker for renal failure and tubular injury. 
     SEQ ID NO:23 (Kininogen) levels generally decreased within the first 1-2 days of dichromate treatment, and recovered (increased) during later time points ( FIG.  3 B ). These data illustrate that decreased SEQ ID NO:23 (Kininogen) levels provide a marker for renal failure and tubular injury. 
     SEQ ID NO:25 (Inter-Alpha Inhibitor H4 (ITIH4)) levels generally decreased by the day 2 of dichromate treatment, and recovered (increased) after day 2 ( FIG.  3 C ). These data illustrate that altered SEQ ID NO:25 (Inter-Alpha Inhibitor H4 (ITIH4)) levels provide a marker for renal failure and tubular injury. 
     In addition, the invention is not intended to be limited to the disclosed embodiments of the invention. It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.