Patent Publication Number: US-2023144446-A1

Title: Blood-based diagnostic assays for alzheimer&#39;s disease

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of priority to U.S. Provisional Pat. Application No. 63/225,423, filed on Jul. 23, 2021, the entire contents of which is incorporated herein by reference in its entirety. 
     REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY 
     The specification further incorporates by reference a concurrently-filed sequence listing submitted electronically via EFS-Web as a file named “SequenceListing.xml”, created on Jul. 22, 2022. The sequence listing contained in this document is part of the specification and is herein incorporated by reference in its entirety. 
    
    
     TECHNICAL FIELD 
     The invention relates to sets of biomarkers and methods of use thereof for diagnosing, staging, treating, and assessing the response of a treatment for neurocognitive disorders characterised by tau toxicity, such as Alzheimer’s disease. 
     BACKGROUND 
     Alzheimer’s disease (AD) represents one of the greatest health care burdens, with 35 million affected individuals worldwide, a population estimated to increase to 115 million by 2050. [Wimo, Alzheimer’s Disease International World Report 2010. The Global Economic Impact of Dementia, Alzheimer’s Disease International (2010).] AD is a devastating dementia that first presents as progressive memory loss and later can include neuropsychiatric symptoms such as depression, paranoia, agitation and even aggression. Currently, available AD treatment is limited to cognitive enhancers with limited and short-lived efficacy. 
     Previously, diagnosis of AD could only be confirmed at autopsy by the presence of amyloid deposits and neurofibrillary tangles (NFTs) containing the microtubule-associated protein tau. Current clinical diagnoses of AD satisfy the DSM-IV TR and the NINCDS-ADRDA Work Group criteria for probable AD in McKhann et al.,  Neurology  34(7):939-944 (1984). Initial diagnostic criteria based mostly on subjective assessments set out in McKhann et al., above, require that the presence of cognitive impairment and a suspected dementia syndrome be confirmed by neuropsychological testing for a clinical diagnosis of possible or probable AD; although they need histopathologic confirmation (microscopic examination of brain tissue) for the definitive diagnosis. 
     The criteria specify eight cognitive domains that can be impaired in AD. Those cognitive domains are: memory, language, perceptual skills, attention, constructive abilities, orientation, problem solving and functional abilities. There are no motor, sensory, or coordination deficits early in the disease. These criteria have shown good reliability and validity, and are those used herein as the basis for assertion of clinical diagnosis of AD. 
     The diagnosis could not heretofore be determined by laboratory assays. Such assays are important primarily in identifying other possible causes of dementia that should be excluded before the diagnosis of Alzheimer’s disease can be made with confidence. Neuropsychological tests provide confirmatory evidence of the diagnosis of dementia and help to assess the course and response to therapy. The criteria proposed by McKhann et al., above, are intended to serve as a guide for the diagnosis of probable, possible, and definite Alzheimer’s disease; these criteria will likely be revised as more definitive information become available. 
     Diagnostic criteria have more recently been refined to include the prodromal phase (early symptoms that occur before the full-blown symptoms of the disease are manifest) termed “Mild Cognitive Impairment (MCI) due to AD.” This new diagnosis reflects a desire to treat the disease earlier because the neuropathology is estimated to start 10 years prior to appearance of symptoms. [Trojanowski et al.,  Alzheimers Dement  6, 230-238 (2010)]. Clinical trials of potential disease-modifying treatments have been hugely disappointing, possibly in part because even an “early-stage” patient already has a massive amyloid-beta (Aβ) burden and substantial pathologies with significant synaptic defects and inflammation. 
     According to Petersen et al.,  Arch Neurol  56(3):303-308 (1999), the primary distinction between control subjects and subjects with MCI is in the area of memory, whereas other cognitive functions are comparable. However, when the subjects with MCI were compared with the patients with very mild AD, memory performance was similar, but patients with AD were more impaired in other cognitive domains as well. Longitudinal performance demonstrated that the subjects with MCI declined at a rate greater than that of the controls but less rapidly than the patients with mild AD. 
     Patients that meet the criteria for MCI can be differentiated from healthy control subjects and those with very mild AD. They appear to constitute a clinical entity that can be characteri zed for treatment interventions. 
     Amyloid-beta (Aβ) is a peptide 39-42 amino acid residues in length that is generated in vivo by specific, proteolytic cleavage of the amyloid precursor protein (APP) by β- and γsecretases. Aβ 42  comprises residues 677-713 of the APP protein, which is itself a 770-residue transmembrane protein having the designation P05067 in the UniProtKB/Swiss-Prot system. Aβ, and in particular the Aβ 42 , is commonly believed to be the principal causative agent in AD, although its mechanism underlying AD neuropathologies is debated. 
     Until recently, only a symptom-based assay as discussed in McKhann et al., above, was available diagnosing the presence of Alzheimer’ s disease in a living patient. More recently, beginning in April of 2012 with Eli Lilly’s Amyvid®, and later with approval of GE Healthcare’s Vizamyl® and Piramal Imaging’s Neuraceq ®, PET scanning technology has been used to assay for AD in a living human. The intravenous–infused, radiolabeled positronemitting compound binds to Aβ in brain plaques. 
     Although accurate, PET scanning assays are inconvenient for patients in that the patients have to place their heads in a relatively confined space within a scintillation detector and should remain relatively motionless. PET scanning assays are also costly, particularly as compared to a more usual blood test that one receives in which a few milliliters of blood is taken to provide for as many as 40 different assays that unfortunately do not yet commercially include a test for AD. 
     Discovered 45 years ago as the first non-muscle actin-binding protein [Hartwig et al.,  J Biol Chem  250:5696-5705 (1975); Wang et al.,  Proc Natl Acad Sci USA  72:4483-4486 (1975)], filamins [FLNs] are a family of cytoskeletal proteins - filamins A (FLNA) and B, but not C - that are expressed in non-muscle cells. Human FLNA is given the identifier P21333 in the UniProtKB/Swiss-Prot data base, and contains a sequence of 2647 amino acid residues (about 280 kDa). This protein is also sometimes referred to in the art as actin-binding protein (ABP-280). [Gorlin et al.,  J Cell Biol  111: 1089-1105 (1990).] 
     The FLNA protein anchors various transmembrane proteins to the actin cytoskeleton and serves as a scaffold for a wide range of cytoplasmic signalling proteins. Filamins are essential for mammalian cell locomotion and act as interfaces for protein-protein interaction [van der Flier et al.,  Biochim Biophys Acta  1538:99-117 (2001)]. Besides its role in cell motility, FLNA is increasingly found to regulate cell signalling by interacting with a variety of receptors and signalling molecules. [Stossel et al.,  Nat Rev Mol Cell Biol  2:138-145 (2001); Feng et al.,  Nat Cell Biol  6:1034-1038 (2004)). 
     The FLNA protein consists of an N-terminal actin-binding domain (ABD) and a rod-like domain of 24 immunoglobulin-like repeat domains (IgFLNa’s) (each about 96-amino acid residues long and numbered from the N-terminus), interrupted by two 30 \-amino acid residue flexible loops or hinges. The IgFLNa’s are numbered 1 through 24, beginning near the N-terminus and ending near the C-terminus. The loop designated H1 is between repeats 15 and 16, and the loop designated H2 is located between repeats 23 and 24 [Gorlin et al.,  J Cell Biol  111:1089-1105 (1990); van der Flier et al.,  Biochim Biophys Acta  1538:99-117 (2001)]. 
     H1 and H2 can be cleaved by calpains and caspases [Gorlin et al.,  J Cell Biol  111:1089-1105 (1990); Browne et al.,  J Biol Chem  275:39262-39266 (2000)]. Cleavage at H1 occurs between ammo acid residues 1762 and 1764, and results in an about 170 kDa fragment consisting of the ABD and repeats 1-15 (IgFLNa-1-15), plus an about 110 kDa polypeptide fragment consisting of repeats 16-24 (IgFLNa-16-24). 
     It is noted that the UniProtKB/Swiss-Prot data base lists a C-terminus for repeat 15 at position 1740 and a N-terminus for repeat 16 as being located at amino acid residue position 1779. On the other hand, Gorlin et al., above, place the calpain cleavage site between residues 1762 and 1764, whereas Garcia et al.,  Arch Biochem Biophys  446: 140-150 (2006) places that site between residues 1761 and 1762. Similarly, Gorlin et al., above, write that earlier authors (Hartwig et al.,  J Cell Biol  87:841-848 (1980)] reported the full length FLNA molecule to have a molecular weight of 270 kDa at page 1089 and thereafter as having a 280 kDa protein at page 1093. 
     Garcia et al. above, reports that calpain cleaves full length FLNA into polypeptide fragments of 180, 100, 90 and 10 kDa, whereas Bedolla et al.,  Clin Cancer Res  15(3):788-796(2009) report proteolysis fragments of 170, 110 and a 90 kDa fragment cleaved from the 110 kDa fragment. Browne et al., above, reported that the cytotoxic T lymphocyte protease granzyme B, grB, cleaves filamin in concert with the lytic protein perforin, and that filamin is also cleaved in a caspase-dependent manner following ligation to a Fas receptor. Western blots of dying Jurkat cell lysates identified two caspase cleavage polypeptides from the C-terminal region of FLNA having masses of about 110 and 95 kDa were identified. Purified grB cleaved filamin into several polypeptides including those having masses of about 205,200 and 110 kDa. Polyclonal rabbit antibodies raised to a fusion protein containing 476 amino acid residues from the C-terminal region of FLNA (positions 2172-2647) were used. Umeda et al. J Biochem 130:535-542 (2001) found somewhat similar results (C-terminal 135, 120 and 110 kDa polypeptide fragments) in U937 monoblastic leukemia and Jurkat human T lymphoblastic cells for proteolysis by caspase-3. 
     It is noted that IgFLNa-16-24 is said to have a mass of about 110 kDa in Loy et al.,  Proc Natl Acad Sci , USA, 100(8):4562-4567 (2003). That about 110 kDa polypeptide (IgFLNa-16-24) is further cleaved at H2 by calpain with a longer digestion time to yield an about 90 kDa, fragment that contains repeats 16-23 (IgFLNa-16-23) [Gorlin et al.,  J Cell Biol  111 :-1089-1105 (1990); van der Flier et al.,  Biochim Biophys Acta  1538:99-117 (2001)]. 
     Because of the differences in residue positions and some molecular weights for the full length FLNA and its proteolysis fragments reported in the art as discussed above, the full length FLNA molecule and the smaller FLNA cleavage product as having molecular weights of “about” 280 kDa and “about” 90 kDa, respectively. 
     FLNA promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. Filamin A is dimerized through the carboxy-terminal repeat (repeat 24) near the transmembrane regions, providing an intracellular V-sliaped structure that is critical for function. 
     Each v-shaped FLNA dimer has two antiparallel self-bound domains 24 forming the apex of the “v”, and the remaining domains stretched out much like beads on a string with each of their N-terminal ABD portions bound to an actin molecule. More recently, it has been reported that C-terminus of the ABD, rod segment 1 (IgFLNa-1-15), forms an extended linear structure without obvious inter-domain interactions. Rod segment 2 (IgFLNa-16-23) assumes a compact structure due to multiple inter-domain interactions in which domains 16-17, 18-19 and 20-21 form paired structures. [Heikkinen et al.,  J Biol Chem , 284:25450-1-5458 (2009); Lad et al.,  EMBOJ , 26:3993--4004 (2007).] 
     Proteolysis of FLNA is regulated in part by its phosphorylation on Ser 2152 (S2152) in repeat 20 (IgFLNa-20), which is reported to render the full-length protein stable and resistant to cleavage. [Gorlin et al.,  J Cell Biol  111: 1089–1105 (1990); Garcia et al.,  Arch Biochem Biophys  446:140–150 (2006); and Chen et al.,  J Biol Chem  264(24):14282-14289 (1989).] 
     Loy et al.,  Proc Natl Acad Sci , USA, 100(8):4562-4567 (2003), report that a H1 cleavage product containing repeats 16-24 and having a molecular weight of about 100 kDa colocalized with the androgen receptor to the nucleus in prostate cancer cells. Those workers noted that FLNA is generally regarded as a cytoplasmic architectural molecule, and characterized their finding of an additional function of its about 100 kDa polypeptide as is produced by calpain cleavage as a nuclear regulator of the androgen receptor to be “entirely unexpected” (at page 4565). 
     The about 100 kDa FLNA fragment found in a cellular nucleus is not phosphorylated on S2152. Indeed, phosphorylation on S2152 is reported to block the cleavage of full length FLNA by calpain in a prostate cancer line and in platelets. [Garcia et al.,  Arch Biochem Biophys  446:140-150 (2006); and Chen et al.,  J Biol Chem  264(24):14282-14289 (1989)]. 
     Wang et al.,  Oncogene  26:6061-6070 (2007), showed that nuclear localization of FLNA correlates with hormone-dependence in prostate cancer. The non-phosphorylated about 90 kDa fragment (IgFLNa-16-23) migrates to the nucleus of hormone-naive cells in the androgen-dependent form of the disease. Contrarily, in hormone-refractory androgenindependent prostate tumor cells, FLNA was phosphorylated, preventing its cleavage and nuclear translocation. Those authors and co-workers subsequently showed that not only does prostate cancer metastasis correlate with cytoplasmic localization of FLNA, but that metastasis can be prevented by cleavage and subsequent nuclear translocation of the phosphorylated protein. [Bedolla et al.,  Clin Cancer Res.  15(3):788–796 (2009).] 
     As a key regulator of the cytoskeleton network, FLNA interacts with many proteins involved in cancer metastasis, [Yue et al.,  Cell &amp; Biosci  3:7 (2013)] as well as in many other conditions. Thus, Nakamura et al.,  Cell Adh Migr.  5(2):160–169 (2011), discuss the history of research concerning FLNA and note that the protein serves as a scaffold for over 90 binding partners including channels, receptors, intracellular signalling molecules and transcription factors. 
     FLNA also has been implicated in tumor progression. FLNA knockout mice show reduced oncogenic properties of K-Ras, including the downstream activation of ERK and Akt. [Nallapalli et al.,  Mol Cancer  11:50 (2012).] Many different cancers show high levels of FLNA expression in contrast to low FLNA levels in corresponding normal tissue, including colorectal and pancreatic cancers, [Uhlen et al.,  Mol Cell Proteomics  4:1920-1932 (2005)] and glioblastoma [Sun et al.,  Cancer Cell  9:287-300 (2006)). 
     Inhibition of FLNA expression sensitizes cancer cells to both cisplatin and radiation [Sun et al.,  Cancer Cell  9:287-300 (2006)], and FLNA deficiency in cancer cells similarly sensitizes them to chemotherapeutic agents [Yue et al.,  DNA Repair (Amst)  11:192-200 (2012)] and radiation [Yue et al.,  Cancer Res  69:7978-7985 (2009); Yuan et al.,  J Biol Chem  276:48318-48324 (2001)]. On the other hand, Jiang et al.,  Int. J. Biol. Sci.  9:67-77 (2013) report that inhibition of filamin A expression leads to reduced metastasis in nude mice implanted with melanoma and breast cancer cells. 
     Phosphorylation has become recognized as a global regulator of cellular activity, and abnormal phosphorylation is implicated in a host of human diseases, particularly cancers. Phosphorylation of a protein involves the enzymatically-mediated replacement of an amino acid side chain hydroxyl of one or more serine, threonine or tyrosine residues with a phosphate group (-OPO 3   -2 ). 
     Phosphorylation and its reverse reaction, dephosphorylation, occur via the actions of two key enzyme types. Protein kinases phosphorylate proteins by transferring a phosphate group from a nucleotide triphosphate such as adenosine triphosphate (ATP) or guanosine triphosphate (GTP) to their target protein. This process is balanced by the action of protein phosphatases, which can subsequently remove the phosphate group. 
     The amount of phosphate that is bonded to a protein at a particular time is therefore determined by the relative activities of the particular one or more associated kinase and phosphatase enzymes specific to that protein and to the particular amino acid residue(s) undergoing phosphorylation/ dephosphorylation. If the phosphorylated protein is an enzyme, phosphorylation and dephosphorylation can impact its enzymatic activity, essentially acting like a switch, turning it on and off in a regulated manner. Phosphorylation can similarly regulate non-enzymatic protein-protein interactions by facilitation of binding to a partner protein. 
     Protein phosphorylation can have a vital role in intracellular signal transduction. Many of the proteins that make up a signaling pathway are kinases, from the tyrosine kinase receptors at the cell surface to downstream effector proteins, many of which are serine/threonine kinases. 
     FLNA is phosphorylated at a number of positions in its protein sequence in both normal and in diseased cells such as cancer cells. For example, the enzyme PAK1 (EC 2.7.11.1) is a protein kinase of the STE20 family that regulates cell motility and morphology. FLNA phosphorylation at position 2152 by PAK1 is required for PAK1-mediated actin cytoskeleton reorganization and for PAK1-mediated membrane ruffling. [Vadlamudi et al.,  Nat. Cell Biol.  4:681-690 (2002); Woo et al.,  Mol Cell Biol.  24(7):3025–3035 (2004).] Cyclin Bl/Cdkl (EC:2.7.11.22; EC:2.7.11.23) phosphorylates serine 1436 in vitro in FLNA-dependent actin remodeling. [Cukier et al., FEBS Letters 581(8):1661-1672 (2007).] 
     The UniProtKB/Swiss-Prot data base entry for human FLNA (No. P21333) lists published reports of the following amino acid residue positions as being phosphorylated under different circumstances: 11, 1081, 1084, 1089, 1286, 1338, 1459, 1533, 1630, 1734, 2053, 2152, 2158, 2284, 2327, 2336, 2414, and 2510. Further, polyclonal and monoclonal antibodies are commercially available from one or more of Abgent, Inc. (San Diego, CA), Abcam® Inc. (Beverly, MA), Bioss, Inc. (Woburn, MA), and GeneTex, Inc. (Irvine, CA) that immunoreact with FLNA that is phosphorylated (phospho-FLNA) at serine-1083, tyrosine-1046, serine-1458, serine-2152, and serine-2522. 
     The 90 kDa FLNA fragment that can localize to the nucleus and interact with transcription factors includes the serine-2152 residue that can be phosphorylated. However, that previously discussed nucleus-localized about 90 kDa FLNA fragment is free of phosphorylation at serine-2152. Indeed, phosphorylation of FLNA at serine-2152 (“pS2152” FLNA) has been reported to protect FLNA from proteolysis to form the about 90 kDa fragment [Garcia et al.,  Arch Biochem Biophys  446: 140-150 (2006); Gorlin et al.,  J Cell Biol  111:1089-1105 (1990); and Chen et al.,  J Biol Chem  264(24):14282-14289 (1989)]. 
     It has been shown that the toxic signalling of amyloid-β42 (Aβ42) by the α7~ nicotinic acetylcholine receptor (α7nAChR), which results in tau phosphorylation and formation of neurofibrillary tangles associated with Alzheimer’s disease, requires the recruitment of the scaffolding protein FLNA to activate TLR4 through CD14, Wang et al.,  J. Neurosci.  32(29):9773-9784 (Jul. 18, 2012). That paper also showed that PTl~125 (simufilam) provides an anti-inflammatory effect by similarly reducing FLNA association with toll-like receptor 4 (TLR4) and preventing cytokine release.” 
     BRIEF SUMMARY OF THE INVENTION 
     Scaffolding protein FLNA is recognized as having an association with the development of Alzheimer’s disease through its involvement of Aβ42 signalling through α7nAChR, and the abnormally folded form of FLNA believed to be the linking agent in this process is the target of PTI-125 (simufilam), a small molecule antagonist of FLNA currently being investigated for use in treatment of AD. Interestingly, FLNA is also strongly expressed in platelets where it is thought to regulate normal platelet functions, and mutations in FLNA can cause platelet-related disorders. Platelets also express functional α7nAChR (Schedel et al.,  ArteriosclThrom, Vas,  31:928-934 (2011.)). There have also been reports that platelets can shuttle Aβ42 to the brain (Catricala et al.,  ImmunAgeing 9:20 (2012). FLNA is also known to be processed in platelets during activation [Buitrago et al.,bioRxiv 307397] and this can assist in creation of abnormally folded forms of FLNA able to bind Aβ42 and/or α7nAChR both within platelets and in the brain following platelet lysis. 
     Current research has failed to produce viable diagnostic assays based on FLNA. Indeed, current diagnostic methods for Alzheimer’s disease typically require tissue samples (e.g., for post-mortem confirmation) or cerebrospinal fluid obtained via invasive surgical procedures. The present disclosure addresses these and other shortcomings associated with current assays by providing new blood-based assays and methods that can be used to diagnose AD and other tauopathies, as well as kits and reagents related to the same based on novel biomarkers (e.g., FLNA having a particular phosphorylation profile) or fragments thereof, for use in methods for diagnosing, staging, treating and assessing the response of a treatment for neurocognitive disorders such as Alzheimer’s disease. In addition, the present invention provides novel targets for the development of new therapies against tauopathies or for repurposing existing therapies not originally designed for the treatment of neurocognitive disorders such as tauopathies. 
     In a first general aspect, the present disclosure provides a biomarker panel comprising one or more phosphorylated peptides obtained from in vitro digestion of Filamin A (SEQ ID NO: 1). In some aspects, the FLNA is contained within or obtained from a biological fluid or tissue sample taken from a subject suspected of having a neurological disease (e.g., Alzheimer’s disease or another tauopathy). For the sake of brevity, example sequences are given based on digestion with the proteases trypsin, GluC and AspN. The skilled practitioner would understand that digestion with other proteases generate alternative peptide sequences comprising the same phosphorylated residues and all such alternatives are encompassed within the present specification. 
     In some aspects, the phosphorylated peptide is phosphorylated at residue serine 2152 of SEQ ID NO: 1. In some aspects, the phosphorylated peptide has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 (i.e., a fragment of full-length FLNA). In some aspects, the biomarker panel can comprise a plurality of phosphorylated fragments of FLNA, e.g., two or more phosphorylated peptides, each comprising a fragment of FLNA and having the amino acid sequence of any one of SEQ ID NOs: 2-5, wherein at least one of the two or more phosphorylated peptides is phosphorylated at a position corresponding to serine 2152 of SEQ ID NO: 1 (full-length FLNA). 
     In some aspects, the phosphorylated peptide is phosphorylated at residue serine 2143 of SEQ ID NO: 1. In some aspects, the phosphorylated peptide has the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:9 (i.e., a fragment of full-length FLNA). In some aspects, the biomarker panel can comprise a plurality of phosphorylated fragments of FLNA, e.g., two or more phosphorylated peptides, each comprising a fragment of FLNA and having the amino acid sequence of SEQ ID NOs: 6-9, wherein at least one of the two or more phosphorylated peptides is phosphorylated at a position corresponding to serine 2143 of SEQ ID NO: 1 (full-length FLNA). 
     In some aspects, the phosphorylated peptide is phosphorylated at residue serine 2180 of SEQ ID NO: 1. In some aspects, the phosphorylated peptide has the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12 (i.e., a fragment of full-length FLNA). In some aspects, the biomarker panel cancomprise a plurality of phosphorylated fragments of FLNA, e.g., two or more phosphorylated peptides, each comprising a fragment of FLNA and having the amino acid sequence of SEQ ID NOs: 10-12, wherein the two or more phosphorylated peptides are phosphorylated at a position corresponding to residue serine 2180 of SEQ ID NO: 1 (full-length FLNA). 
     In some aspects, the phosphorylated peptide is phosphorylated at residue serine 1459 of SEQ ID NO: 1. In some aspects, the phosphorylated peptide has the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 (i.e., a fragment of full-length FLNA). In some aspects, the biomarker panel can comprise a plurality of phosphorylated fragments of FLNA, e.g., two or more phosphorylated peptides, each comprising a fragment of FLNA and having the amino acid sequence of SEQ ID NOs: 13-15, wherein the two or more phosphorylated peptides are phosphorylated at a position corresponding to residue serine 1459 of SEQ ID NO: 1 (full-length FLNA). 
     In some aspects, the biomarker panel comprises a plurality of phosphorylated peptides, wherein each phosphorylated peptide is a fragment of FLNA (SEQ ID NO: 1) and comprises a phosphorylation site at a position corresponding to serine 1459, 2143, 2152, and/or 2180 of full-length FLNA (SEQ ID NO:1). For example, the biomarker panel can comprise a plurality of phosphorylated peptides, each having the sequence of SEQ ID Nos: 2-15 and being phosphorylated at a position corresponding to serine 1459, 2143, 2152, and/or 2180 of full-length FLNA (SEQ ID NO: 1). In some aspects, the biomarker panel can comprise one or more phosphorylated peptides which comprise a fragment of full-length FLNA that is phosphorylated at two or more of the above-identified phosphorylation sites (e.g., at serine 1459, 2143, 2152, and/or 2180 of full-length FLNA). 
     In some aspects, the biomarker panel comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different phosphorylated peptides, each being phosphorylated at a site corresponding to serine residue 1459, 2143, 2152 and/or 2180 of full-length FLNA (SEQ ID NO: 1). 
     In some aspects, the biomarker panel further comprises one or more peptides listed in Table 4, which can be obtained from in vitro digestion of Filamin A using a protease or combination of proteases, wherein each of the one or more peptides comprises an amino acid sequence present in SEQ ID NO:1. In some aspects, the peptide is proteotypic for FLNA. 
     In a second general aspect, the disclosure provides a panel of biomarkers, comprising: (i) one or more peptides obtained from in vitro digestion of Filamin A using a protease or combination of proteases, wherein each of the one or more peptides comprises an amino acid sequence present in SEQ ID NO:1; and (ii) one or more peptides obtained from in vitro digestion of one or more, optionally two or more of the proteins listed in Table 1. In some aspects, the one or more peptides are obtained from in vitro digestion with one or a combination of proteases selected from trypsin, GluC, ArgC, AspN, and/or chymotrypsin. In some aspects, the Filamin A and one or more proteins listed in Table 1 are contained within a biological fluid or tissue sample obtained from a subject suspected of having a neurological disease. In some aspects, the neurological disease is Alzheimer’s disease or another tauopathy. In some aspects, the one or more peptides obtained from in vitro digestion of Filamin A comprise one or more phosphorylation sites. In some aspects, the one or more phosphorylation sites correspond to serine residue 2143, 2152 and/or 2180 of full-length FLNA (SEQ ID NO: 1). 
     In a third general aspect, the disclosure provides a panel of biomarkers, comprising: (i) one or more peptides obtained from in vitro digestion of Filamin A using a protease or combination of proteases, wherein each of the one or more peptides comprises an amino acid sequence present in SEQ ID NO: 1; and (ii) one or more peptides obtained from in vitro digestion of one or more, optionally two or more, of the proteins listed in Table 2. In some aspects, the one or more peptides are obtained from in vitro digestion with one or a combination of proteases selected from trypsin, GluC, ArgC, AspN, and/or chymotrypsin. In some aspects, the Filamin A and one or more proteins listed in Table 2 are contained within a biological fluid or tissue sample obtained from a subject suspected of having a neurological disease. In some aspects, the neurological disease is Alzheimer’s disease or another tauopathy. In some aspects, the one or more peptides obtained from in vitro digestion of Filamin A comprise one or more phosphorylation sites. In some aspects, the one or more phosphorylation sites correspond to serine residue 2143, 2152 and/or 2180 of full-length FLNA (SEQ ID NO: 1). 
     In a fourth general aspect, the disclosure provides a panel of biomarkers, comprising: (i) one or more peptides obtained from in vitro digestion of Filamin A using a protease or combination of proteases, wherein each of the one or more peptides comprises an amino acid sequence present in SEQ ID NO: 1; and (ii) one or more peptides obtained from in vitro digestion of one or more, optionally two or more, of the proteins listed in Table 3. In some aspects, the one or more peptides are obtained from in vitro digestion with one or a combination of proteases selected from trypsin, GluC, ArgC, AspN, and/or chymotrypsin. In some aspects, the Filamin A and one or more proteins listed in Table 3 are contained within a biological fluid or tissue sample obtained from a subject suspected of having a neurological disease. In some aspects, the neurological disease is Alzheimer’s disease or another tauopathy. In some aspects, the one or more peptides obtained from in vitro digestion of Filamin A comprise one or more phosphorylation sites. In some aspects, the one or more phosphorylation sites correspond to serine residue 2143, 2152 and/or 2180 of full-length FLNA (SEQ ID NO: 1). 
     In a fifth general aspect, the disclosure provides a panel of biomarkers, comprising: (i) one or more peptides obtained from in vitro digestion of Filamin A using a protease or combination of proteases, wherein each of the one or more peptides comprises an amino acid sequence present in SEQ ID NO: 1; and (ii) one or more peptides obtained from in vitro digestion of one or more, optionally two or more of the proteins listed in Tables 1, 2, 3, or 4. In some aspects, the one or more peptides are obtained from in vitro digestion with one or a combination of proteases selected from trypsin, GluC, ArgC, AspN, and/or chymotrypsin. In some aspects, the Filamin A and one or more proteins listed in Table 1 are contained within a biological fluid or tissue sample obtained from a subject suspected of having a neurological disease. In some aspects, the neurological disease is Alzheimer’s disease or another tauopathy. 
     In a sixth general aspect, the disclosure provides a panel of biomarkers, comprising: one or more synthetic peptides, wherein one or more of the synthetic peptides are enriched with heavy isotopes of H, C, N, O and/or S. In some aspects, one or more of the synthetic peptides comprises an amino acid sequence present in in Filamin A (SEQ ID NO: 1); or present in one of the proteins listed in Tables 1, 2, 3, or 4. In some aspects, one or more of the synthetic peptides comprises an amino acid sequence corresponding to the sequence of a peptide obtained from in vitro digestion of SEQ ID NO: 1 or of one of the proteins listed in Tables 1, 2, 3, or 4, using one or a combination of proteases selected from trypsin, GluC, ArgC, AspN, and/or chymotrypsin. 
     In a seventh general aspect, the disclosure provides a method for the diagnosis/prognosis of a neurological disorder, comprising: obtaining a bodily fluid or tissue sample from a subject (optionally, a subject suspected of having a neurological disorder such as Alzhiemer’s disease); digesting one or more proteinaceous materials (e.g., proteins and/or polypeptides) in the bodily fluid or tissue sample with one or more proteases (e.g., trypsin, GluC, ArgC, AspN, and/or chymotrypsin); and detecting and/or measuring the level of one or more peptides produced from the digestion, using mass spectrometry. In some aspects, each of the one or more peptides comprises an amino acid sequence corresponding to the sequence of a peptide obtained from in vitro digestion of Filamin A (SEQ ID NO: 1) or of one of the proteins listed in Tables 1, 2, or 3. 
     In an eighth general aspect, the disclosure provides a method for monitoring the progression of a neurological disorder, comprising: (a) obtaining a bodily fluid or tissue sample from a subject (optionally, a subject suspected of having a neurological disorder such as Alzhiemer’s disease) at a first time point; (b) digesting one or more proteins in the bodily fluid or tissue sample with one or more proteases (e.g., trypsin, GluC, ArgC, AspN, and/or chymotrypsin); (c) detecting and/or measuring the level of one or more peptides produced from the digestion, using mass spectrometry; and (d) repeating steps (a)-(c) at a second time point. In some aspects, the method further comprises step (e) of determining whether a therapeutic treatment administered to the subject is effective in treating the neurological disorder based upon the detected and/or measured levels of the one or more peptides of step (c). In some aspects, each of the one or more peptides comprises an amino acid sequence corresponding to the sequence of a peptide obtained from in vitro digestion of Filamin A (SEQ ID NO: 1) or of one of the proteins listed in Tables 1, 2, or 3. 
     In a ninth general aspect, the disclosure provides a method for diagnosing or monitoring the progression of a neurological disorder (such as Alzheimer’s disease or another tauopathy), comprising: obtaining a bodily fluid or tissue sample from the subject; digesting one or more proteins in the bodily fluid or tissue sample with one or more proteases (e.g., trypsin, GluC, ArgC, AspN, and/or chymotrypsin); and measuring the level if any of one or more phosphorylated peptide fragments produced from the digestion of Filamin A (SEQ ID NO:1). In some aspects, the one or more fragments comprise a phosphorylated serine at a position corresponding to serine 2152 and/or 2143 of Filamin A (SEQ ID NO:1). 
     In some aspects, the method further comprises determining a ratio of two or more of the above peptide fragments. In some aspects, the ratio comprises a ratio of fragments that are phosphorylated at a position corresponding to serine 2143 of Filamin A (SEQ ID NO:1) versus fragments that are phosphorylated at a position corresponding to serine 2152 of Filamin A (SEQ ID NO:1). In some aspects, the ratio is determined using two or more peptides that each comprise a sequence of SEQ ID Nos: 2-9. In some aspects, the ratio comprises a ratio of fragments that are phosphorylated at a position corresponding to serine 2143 of Filamin A (SEQ ID NO: 1) versus fragments which are phosphorylated at a position corresponding to serine 2152 of Filamin A (SEQ ID NO:1); wherein a ratio of &lt;5 indicates that the subject does not have Alzheimer’s disease and a ratio of &gt;10 indicates that the subject has Alzheirner’s disease. 
     In a tenth general aspect, the disclosure provides a method for diagnosing or monitoring the progression of a neurological disorder (such as Alzheimer’s disease or another tauopathy), comprising: obtaining a bodily fluid or tissue sample from the subject; digesting one or more proteins in the bodily fluid or tissue sample with one or more proteases (e.g., trypsin, GluC, ArgC, AspN, and/or chymotrypsin); and measuring the level of any of one or more phosphorylated peptides produced from the digestion of Filamin A (SEQ ID NO: 1) and one or more peptides derived from Integrin alpha-IIb, Integrin beta-3, and/or a Linker for activation of T-cells family member 1, wherein the presence of Integrin alpha-IIb, Integrin beta-3, and/or Linker for activation of T-cells family member 1, indicates ex vivo or artefactual platelet activation. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1    shows a representation of the TMTcalibrator™ mass spectrometry analysis workflow. After depletion of the top 14 abundant proteins, plasma samples were digested and labelled with TMTpro™ reagents. In parallel, brain lysates were also digested and labelled with TMTpro™ reagents. Plasma and brain lysate digests were then mixed and a small aliquot analysed for total proten expression by tandem mass spectrometry (LC-MS/MS). The phosphopeptide fraction was enriched from the remaining mixture and analysed by LC-MS/MS. Data analysis identified peptide sequences and relative abundances based on TMTpro™ reporter ions and linear modelling used to identify features showing differential expression in AD and Control samples. 
         FIG.  2    shows a histogram of the expression profile for FLNA in 6 AD plasma samples prepared on HistopaqueⓇ-1077 and 6 plasma samples collected in EDTA tubes. Values are expressed as log2 ratios relative to the reference brain lysate channel. 
         FIG.  3    shows a histogram of the expression profile for the tryptic peptide APsVANVGSHCDLSLK phosphorylated at serine 2152 of FLN.A (SEQ ID NO: 1) in 6 AD plasma samples prepared on HistopaqueⓇ-1077 and 6 plasma samples collected in EDTA tubes. Values are expressed as isotope-corrected TMTpro™ reporter ion intensities. 
         FIG.  4    shows a histogram of the expression profile for the tryptic peptide RAPsVANVGSJCDLSLK phosphorylated at serine 2152 of FLNA (SEQ ID NO: 1) in 6 AD plasma samples prepared on Histopaque®-1077 and 6 plasma samples collected in EDTA tubes. Values are expressed as isotope-corrected TMTpro™ reporter ion intensities. 
         FIG.  5    shows a histogram of the expression profile for the tryptic peptide VKEsITR phosphorylated at serine 2143 of FLNA (SEQ ID NO: 1) in 6 AD plasma samples prepared on HistopaqueⓇ-1077 and 6 plasma samples collected in EDTA tubes. Values are expressed as isotope-corrected TMTpro™ reporter ion intensities. 
         FIG.  6    shows a histogram of the expression profile for the tryptic peptide IPEISIQDMTAQVTsPSGK phosphorylated at serine 2180 of FLNA (SEQ ID NO: 1) in 6 AD plasma samples prepared on HistopaqueⓇ-1077 and 6 plasma samples collected in EDTA tubes. Values are expressed as isotope-corrected TMT’pro™ reporter ion intensities. 
         FIGS.  7 A,  7 B,  7 C, and  7 D ) show four box-and-whisker plots representing the relative expression of the four FLNA phosphopeptides RAPsVANVGSHCDLSLK(SEQ ID NO: 3;  FIG.  7 A ), IPEISIQDMTAQVTsPSGK (SEQ ID NO: 10;  FIG.  7 B ), VKEsITR (SEQ ID NO: 6;  FIG.  7 C ) and CSGPGLsPGMVR (SEQ ID NO: 13;  FIG.  7 D ) in 6 AD plasma samples prepared on Histopaque®-1077 and 6 plasma samples collected in EDTA tubes. The values shown are log2 ratios relative to the reference brain lysate channel. 
         FIGS.  8 A,  8 B, and  8 C  show heatmaps of log2 transformed phosphopeptide expression levels relative to the brain lysate channel in 6 AD plasma samples prepared on HistopaqueⓇ-1077 and 6 plasma samples collected in EDTA tubes. The parent gene names of the regulated phosphopeptides are shown to the right of the figure in each panel.  FIG.  8 A  shows regulated phosphopeptides in AD cases versus old controls.  FIG.  8 B  shows regulated phosphopeptides in AD cases versus young controls.  FIG.  8 C  shows regulated phosphopeptides in AD cases versus all controls. 
         FIGS.  9 A,  9 B, and  9 C  provide histograms showing the expression profile for the tryptic peptides VKE[p]sITRphosphorylated at serine 2143 of FLNA (SEQ ID NO: 1) ( FIG.  9 A ), RAP[p]sVANVGSHCDLSLK phosphorylated at serine 2152 of FLNA (SEQ ID NO: 1) ( FIG.  9 B ), and AP[p]sVANVGSHCDLSLK (SEQ ID NO: 138) phosphorylated at serine 2152 of FLNA (SEQ ID NO: 1) ( FIG.  9 C ); in 6 AD plasma samples prepared on Histopaque®-1077 and 6 plasma samples collected in EDTA tubes. Values are expressed as isotope-corrected TMTpro™ reporter ion intensities. 
         FIGS.  10 A,  10 B,  10 C, and  10 D  show four box-and-whisker plots representing the relative expression of the four FLNA phosphopeptides, AP[p]sVANVGSHCDLSLK (SEQ ID NO: 1;  FIG.  10 A ) RAP[p]sVANVGSHCDLSLK (SEQ ID NO: 3,  FIG.  10 B ), rL[p]tVSSLQESGLk (SEQ ID NO: 186;  FIG.  10 C ), and CSGPGL[p]sPGMVR (SEQ ID NO: 13;  FIG.  10 D ) in 6 AD plasma samples prepared on HistopaqueⓇ-1077 and 6 plasma samples collected in EDTA tubes. The values shown are log2 ratios relative to the reference brain lysate channel. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     For purposes of interpreting this specification, the following definitions and abbreviations will apply unless specified otherwise and whenever appropriate, terms used in the singular will also include the plural and vice versa. 
     The term “biomarker(s)” includes all biologically relevant forms of the protein identified, including post-translational modifications. For example, the biomarker can be present in a glycosylated, phosphorylated, multimeric, fragmented or precursor form. A biomarker fragment can be naturally occurring or, for example, enzymatically generated and still retaining the biologically active function of the full protein. Fragments will typically be at least about 10 amino acids, usually at least about 50 amino acids in length, and can be as long as 300 amino acids in length or longer. 
     The term “canonical sequence” is used herein as to refer to the most prevalent sequence and/or the most similar sequence among orthologous species. In particular, unless otherwise specified, the canonical sequence refers herein to the human sequence. 
     The peptide sequences disclosed herein are represented using the IUPAC single-letter code. Use of lower-case letters indicate a modification as follows: “c” —carbamidomethylated cystine; “m” - oxidized methionine; “n” — de-amidated asparagine; “q” - de-amidated glutamine; and “k” - TMT-labelled lysine. A lower case letter at the N-terminus represents a TMT-modified amino acid. In addition, the symbol “[p]” is used to indicate phosphorylation on the succeeding amino acid, e.g., “[p]s” denotes a phosphorylated serine; “[p]t” denotes a phosphorylated threonine; and “[p]y” denotes a phosphorylated tyrosine. 
     The term “KEGG pathway” refers to a collection of manually drawn pathway maps representing molecular interactions and reaction networks for metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, human diseases and drug development. “KEGG pathways mapping” is the process to map molecular datasets, especially large-scale datasets in genomics, transcriptomics, proteomics, and metabolomics, to the KEGG pathway maps for biological interpretation of higher-level systemic functions; (http://www.genome.jp/kegg/pathway.html). 
     The term “concentration or amount” refers to the relative concentration or amount of biomarker in the sample, for example as determined by LC-MS/MS label free quantification approaches such as area under the curve and spectral counting. 
     The term “comparing” or “compare” or grammatical equivalents thereof, means measuring the relative concentration or amount of a biomarker in a sample relative to other samples (for example protein concentrations or amounts stored in proprietary or public database). 
     The term “reference concentration or amount” refers to, but it is not limited to, protein concentrations or amounts stored in proprietary or public databases. The “reference concentration or amount” can have been obtained from a large screening of patients, or by reference to a known or previously determined correlation between such a determination and clinical information in control patients. For example, the reference values can be determined by comparison to the concentration or amount of the biomarkers in a control subject, for example a healthy person (i.e. without dementia) of similar age and gender as the subject. Alternatively, the reference values are values that can be found in literature such as the A.poE 24 allele presence whereby the presence or absence of the mutations at position 112 and 158 represent the reference to be compared to, or like the levels of total tau (T-tau) &gt;350 ng/L, phospho-tau (P-tau) &gt;80 ng/L and A~42 &lt;530 ng/L in the CSF (Hansson et al.,  Lancet Neural.  5(3):228-234 (2006). In addition, the reference values can have been obtained from the same subject at one or more time points that precede in time the test time point. Such earlier samples can be taken one week or more, one month or more, three months or more, most preferably six months or more before the date of the test time point. In some embodiments, multiple earlier samples can be compared in a longitudinal manner and the slope of change in biomarker expression, if any, can be calculated as a correlate of cognitive change, such as the usually noted decline. 
     The term “control” or as used herein “non AD control” or “non AD subject” refers to a tissue sample or a bodily fluid sample taken from a human or non-human subject that is cognitively normal or diagnosed with or presenting symptoms of a cognitive abnormality but defined, with respect to the existing biochemical tests, as non AD subjects. 
     The terms “selected reaction monitoring”, “SRM” and “MRM” mean a mass spectrometry assay whereby precursor ions of known mass-to-charge ratio representing known biomarkers are preferentially targeted for analysis by tandem mass spectrometry in an ion trap or triple quadrupole mass spectrometer. During the mass spectral analysis, the parent ion is fragmented and the number of selected daughter ions of a second predefined mass-to-charge ratio is counted. Typically, an equivalent precursor ion bearing a predefined number of stable isotope substitutions but otherwise chemically identical to the target ion is included in the method to act as a quantitative internal standard. 
     The terms “parallel reaction monitoring” and “PRM” mean a mass spectrometry assay whereby precursor ions of known mass-to-charge ratio representing known biomarkers are preferentially targeted for analysis by tandem mass spectrometry in an Orbitrap™ mass spectrometer (Thermo Fisher Scientific, Waltham, MA). During the analysis, the parent ion is fragmented and the number of each of the daughter ions is counted. Typically, an equivalent precursor ion bearing a predefined number of stable isotope substitutions but otherwise chemically identical to the target ion is included in the method to act as a quantitative internal standard. 
     The term “proteotypic” means a peptide that is uniquely representative of the protein from which it is derived such that the sequence of amino acid residues in the peptide is not found in any other protein from the same species, apart from other expressed isoforms or splice variants derived from the same gene. 
     The term “phosphopeptide” means a peptide that contains at least one amino acid modified by addition of a phosphate group. 
     The term “isolated”, or grammatical equivalents thereof, means throughout this specification, that the protein, peptide, antibody, polynucleotide or chemical molecule as the case can be, exists in a physical milieu distinct from that in which it can occur in nature. 
     As used herein, the term “subject” includes any human or non-human animal. 
     The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, rodents, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. 
     The term “treat”, “treating”, “treatment”, “prevent”, “preventing” or “prevention”, or grammatical equivalents thereof, includes therapeutic treatments, prophylactic treatments and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses the reduction of the symptoms or underlying risk factors. 
     The term “diagnosis”, or grammatical equivalents thereof, as used herein, includes the provision of any information concerning the existence or non-existence or absence or probability of the disorder in a patient. It further includes the provision of information concerning the type or classification of the disorder or of symptoms that are or can be experienced in connection with it. This can include, for example, diagnosis of the severity of the disorder. The term “diagnosis” encompasses prognosis of the medical course of the disorder, for example its duration, severity and the course of progression from mild cognitive impairment (MCI) to AD or other dementias. 
     The term “staging”, or grammatical equivalents thereof, as used herein, means identifying in a subject the stage of a neurocognitive disorder, in particular AD. For example, AD is characterised by 3 stage or 7 stages, depending on the diagnostic framework used. The Global Dementia Scale is one such measure of global function. It is measured by assessment of severity including cognition and function against a standardised set of severity criteria. 
     The term “efficacy” indicates the capacity for beneficial change of a given intervention (e.g. a drug, medical device, surgical procedure, etc.) If efficacy is established, that intervention is likely to be at least as good as other available interventions, to which it has been compared. The term “efficacy” and “effectiveness” are used herein interchangeably. 
     The term “comprising” indicates that the subject includes all the elements listed, but can, optionally, also include additional, unnamed elements. 
     The term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein, 
     Unless the context dictates otherwise, the definitions of the features/terms set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described herein. 
     The following abbreviations shall be understood as following in the context of the specification: CSF (cerebrospinal fluid); LBD (Lewy body dementia); FTD (fronto-temporal dementia); VaD (vascular dementia); ALS (amyotrophic lateral sclerosis); CJD (Creutzfeldt-Jakob disease); CNS (central nervous system); TMT® (Tandem Mass Tag®); TEAB (Tetra-ethylamonium Bicarbonate); TFA (Trifluoroacetic acid); SDS (Sodium dodecyl sulfate); TCEP (Tris(2-carboxyethyl)phosphine); ACN (Acetonitrile); Da (Dalton); HPLC (High-performance liquid chromatography); FA (Formic acid); LC-MS/MS (Liquid Chromatography with tandem Mass Spectrometry detection); MS (Mass Spectrometry); MS/MS or MS2 (Tandem MS); MS/MS/MS or MS3 (triple MS); PAGE (Polyacrylamide gel electrophoresis); SCX (Strong Cation Exchange); ppm (Parts per million); TiO2 (titanium dioxide); IMAC (iron metal affinity chromatography). 
     In AD pathogenesis, accumulation of the amyloid-6 peptide (AB) interacts with the signalling pathways that regulate the phosphorylation of tau. Hyperphosphorylation of tau disrupts its normal function in regulating axonal transport and leads to the accumulation of neurofibrillary tangles and toxic species of soluble tau. Currently there is no cure for AD. 
     Approved treatments are few and of limited efficacy for AD, serving mostly to slow or delay progression. Identification and development of new therapies for the treatment of AD and other tauopathies are also greatly affected by the lack of effective diagnostic, prognostic and predictive biomarkers and by the lack of new targets for the design of new therapies. Currently, AD can only be definitively diagnosed by brain biopsy or upon autopsy after a patient has died. Clearly, in clinical settings, brain biopsy is rarely performed and diagnosis is still primarily made based on the history of the symptoms and depends on a battery of neurological, psychometric and biochemical tests. 
     These latter tests include assessment of ApoE e4 allele status and measurements of amyloid beta, tau and phospho-tau in cerebrospinal fluid. These present methods, nevertheless, are still unsatisfactory not only for the early diagnosis of AD and other tauopathies, but also for predicting the progression of neurological disease that is important for recruitment of patients for clinical trials, for designing new therapies and for predicting the effectiveness of current and new therapies. Accordingly, the present disclosure provides new blood-based assays and methods that can be used to diagnose AD and other tauopathies and to monitor or predict the progression of these diseases (e.g., to guide treatment decisions). 
     The ideal diagnostic biomarkers should have high specificity for disease versus non-disease and high sensitivity to distinguish between disease types and stages. Prognostic biomarkers should reflect the intensity and severity of the pathological changes and predict their future course from a very early stage of the disease, before degeneration is observed, until advanced stages of the disease. Pharmacodynamic biomarkers should give a reliable indication whether an administered therapy is efficacious based on the changes in the level of disease-related proteins in readily accessible body fluids such as blood, blood products including platelets, serum and most preferably plasma and CSF. It is also desirable that such pharmacodynamics biomarkers can provide guidance to clinicians when to stop treatment or switch to a different therapy. 
     New targets need to be efficacious, safe, meet clinical and commercial needs and, above all, be “druggable.” A “druggable” target is accessible to the putative drug molecule, be that a small molecule or larger biologicals and upon binding, elicit a biological response that can be measured both in vitro and in vivo. In other words, its inhibition or activation results in a therapeutic effect in a disease state. Hence, there remains a need for proteins and/or peptides that can perform with superior sensitivity and/or specificity as biomarkers in the diagnosis, staging, prognostic monitoring and assessment of the effectiveness of treatments for patients with Alzheimer’s disease and other tauopathies and can serve as new targets for the development of new therapies. 
     Filamin A (FLNA) is a widely expressed actin-binding protein that regulates cell morphology and is particularly expressed in structured cells such as neurons, although expression levels are generally higher in other organs such as lung, kidney and muscle (Human Protein Atlas). FLNA can be phosphorylated at a number of serine, threonine and tyrosine residues in vivo, and the parent protein of approximately 280 kDa is processed to form fragments of approximately 110 kDa and 90 kDa. It has previously been shown that all three major isoforms of FLNA can be phosphorylated at serine 2152 and that the relative intensity of staining in Western blot with a pS2152-specific antibody can differentiate patients with Alzheimer’s disease (AD) from cognitively normal controls. 
     Modification of FLNA by proteolytic cleavage and phosphorylation is thought to promote changes in the protein structure and/or conformation in the brain. Other disease-specific factors, yet to be identified, may also render FLNA to have altered conformation(s). FLNA, having an altered conformation, is believed to play a role in a toxic signalling mechanism of AB oligomers. In Alzheimer’s patients, the altered FLNA independently interacts with both the α7-nicotinic acetylcholine receptor (α7nAChR) and toll-like receptor 4 (TLR4). Signalling through a7nAChR is reported to hyperphosphorylate tau and signalling through TLR4 is reported to induce neuroinflammation. 
     In addition, FLNA is a regulator of the actin cytoskeleton, which is important for synaptic function, suggesting another mechanism through which FLNA can contribute to cognitive dysfunction. As such, altered FLNA represents a promising target for new AD therapeutics and is the target of simufilam (PTI-125), a small molecule structural modulator that lessens or prevents the pathological effects of altered FLNA. 
     The use of a pS2152 specific antibody for diagnosis of AD has not yet been validated. In part, this may be due to the potential for the antibody to bind to the unphosphorylated protein, or may reflect the lack of consistency in phosphorylation in human populations. Further to the use of pS2152 antibodies in a Western Blot, where individual isoforms can be easily distinguished on the basis of their molecular weights, the ability to distinguish FLNA isoforms (in which the pS2152 epitope is present in each isoform) in a liquid phase assay such as an ELISA remains a challenge. Furthermore, the widespread expression of FLNA, especially that found in platelets, makes measurement in peripheral fluids prone to potential false-positives. To date, it has not been demonstrated that measuring specific FLNA isoforms or phosphorylations can be used reliably for diagnosis of AD. 
     In view of the limitations of FLNA profiling using antibodies, the present disclosure provides a more specific method of bottom-up mass spectrometry to profile the distribution of FLNA-derived tryptic peptides in the blood plasma of patients with a particular focus on measurement of specific phosphorylation events. Specifically, in some aspects the disclosure provides assays that use the TMTcalibrator™ method (U.S. Pat. No. 10,976,321; [Russel et al.,  Rapid Commun. Mass Spectrom.  31:153—159 (2017)] where isobarically-labelled digests of AD brain tissue lysate are mixed with similarly-labelled plasma samples and wherein the brain lysate digests are present in different concentrations, and their total is in an excess relative to the total concentration of the digests from the plasma channels. By this arrangement, the high concentration of disease-associated proteins in the brain lysates act as a booster for the same proteins present in the plasma, even when these are at concentrations normally below that required for bottom-up mass spectrometry. Using this approach greatly enhances the ability to measure peptides derived from FLNA, and also allows analysis of a much wider group of proteins with common expression in AD brain and plasma.  
     
       
         
          TABLE 1
           
               
               
               
               
               
               
             
               
                 Biomarkers of Group A (Significantly regulated proteins) 
               
               
                 Gene Name 
                 Protein Name 
                 SEQ ID NO: 
                 log2 Fold-Change AD vs Control 
                 pValue 
                 Adjusted pValue 
               
             
            
               
                 ARPC1B 
                 Actin-related protein ⅔ complex subunit 1B 
                 16 
                 2.54 
                 1.16E-09 
                 4.06E-07 
               
               
                 ARPC5 
                 Actin-related protein ⅔ complex subunit 5 
                 17 
                 1.38 
                 4.33E-09 
                 1.33E-06 
               
               
                 ADK 
                 Adenosine kinase 
                 18 
                 2.10 
                 3.95E-06 
                 2.43E-04 
               
               
                 ANXA3 
                 Annexin A3 
                 19 
                 0.98 
                 3.15E-08 
                 6.55E-06 
               
               
                 BIN2 
                 Bridging integrator 2 
                 20 
                 4.22 
                 9.84E-12 
                 4.04E-08 
               
               
                 CALD1 
                 Caldesmon 
                 21 
                 1.83 
                 1.54E-08 
                 3.79E-06 
               
               
                 CALM3 
                 Calmodulin-3 
                 22 
                 1.12 
                 4.60E-07 
                 4.59E-05 
               
               
                 CALR 
                 Calreticulin 
                 23 
                 1.22 
                 1.03E-07 
                 1.39E-05 
               
               
                 CAVIN2 
                 Caveolae-associated protein 2 
                 24 
                 2.04 
                 7.05E-09 
                 1.97E-06 
               
               
                 CLIC1 
                 Chloride intracellular channel protein 1 
                 25 
                 2.09 
                 1.24E-07 
                 1.63E-05 
               
               
                 MOXD1 
                 DBH-like monooxygenase protein 1 
                 26 
                 1.45 
                 3.31E-06 
                 2.17E-04 
               
               
                 DAP 
                 Death-associated protein 1 
                 27 
                 2.06 
                 3.89E-06 
                 2.43E-04 
               
               
                 ERLIN1 
                 Erlin-1 
                 28 
                 0.97 
                 1.58E-06 
                 1.27E-04 
               
               
                 FLNA 
                 Filamin-A 
                 1 
                 1.73 
                 1.36E-10 
                 1.03E-07 
               
               
                 PDE8B 
                 High affinity cAMP-specific and IBMX-insensitive 3’,5’-cyclic phosphodiesterase 8B 
                 29 
                 -1.11 
                 6.60E-08 
                 1.03E-05 
               
               
                 IHH 
                 Indian hedgehog protein 
                 30 
                 -0.97 
                 1.06E-06 
                 9.42E-05 
               
               
                 ITGA2B 
                 Integrin alpha-IIb 
                 31 
                 4.41 
                 7.13E-08 
                 1.07E-05 
               
               
                 ITGB3 
                 Integrin beta-3 
                 32 
                 3.76 
                 2.39E-08 
                 5.26E-06 
               
               
                 LRRFIP2 
                 Leucine-rich repeat flightless-interacting protein 2 
                 33 
                 1.26 
                 2.03E-06 
                 1.47E-04 
               
               
                 SERPINB1 
                 Leukocyte elastase inhibitor 
                 34 
                 1.64 
                 4.38E-08 
                 7.65E-06 
               
               
                 CTSA 
                 Lysosomal protective protein 
                 35 
                 1.24 
                 1.51E-06 
                 1.24E-04 
               
               
                 MANF 
                 Mesencephalic astrocyte-derived neurotrophic factor 
                 36 
                 1.71 
                 9.32E-08 
                 1.35E-05 
               
               
                 MSN 
                 Moesin 
                 37 
                 1.50 
                 1.03E-09 
                 3.92E-07 
               
               
                 MYL6 
                 Myosin light polypeptide 6 
                 38 
                 2.61 
                 5.90E-10 
                 2.47E-07 
               
               
                 MTPN 
                 Myotrophin 
                 39 
                 1.11 
                 4.45E-09 
                 1.33E-06 
               
               
                 PDLIM1 
                 PDZ and LIM domain protein 1 
                 40 
                 2.76 
                 3.47E-10 
                 1.62E-07 
               
               
                 PPL 
                 Periplakin 
                 41 
                 1.04 
                 2.66E-06 
                 1.80E-04 
               
               
                 PLEK 
                 Pleckstrin 
                 42 
                 3.17 
                 1.73E-10 
                 1.03E-07 
               
               
                 PFN1 
                 Profilin-1 
                 43 
                 1.35 
                 1.65E-10 
                 1.03E-07 
               
               
                 PDIA3 
                 Protein disulfide-isomerase A3 
                 44 
                 0.98 
                 1.91E-06 
                 1.43E-04 
               
               
                 S100A12 
                 Protein S100-A12 
                 45 
                 3.68 
                 2.10E-07 
                 2.38E-05 
               
               
                 S100A4 
                 Protein S100-A4 
                 46 
                 3.20 
                 5.70E-07 
                 5.31E-05 
               
               
                 S100A9 
                 Protein S100-A9 
                 47 
                 2.74 
                 2.26E-06 
                 1.58E-04 
               
               
                 ARHGDIB 
                 Rho GDP-dissociation inhibitor 2 
                 48 
                 1.05 
                 1.84E-06 
                 1.41E-04 
               
               
                 SH3BGRL3 
                 SH3 domain-binding glutamic acid-rich-like protein 3 
                 49 
                 2.09 
                 1.90E-08 
                 4.42E-06 
               
               
                 STOM 
                 Stomatin 
                 50 
                 1.46 
                 3.28E-08 
                 6.55E-06 
               
               
                 TLN1 
                 Talin-1 
                 51 
                 1.54 
                 1.72E-10 
                 1.03E-07 
               
               
                 TMSB4X 
                 Thymosin beta-4 
                 52 
                 2.10 
                 4.35E-08 
                 7.65E-06 
               
               
                 TAGLN2 
                 Transgelin-2 
                 53 
                 2.55 
                 1.33E-10 
                 1.03E-07 
               
               
                 TPM3 
                 Tropomyosin alpha-3 chain 
                 54 
                 1.33 
                 1.88E-07 
                 2.25E-05 
               
               
                 TPM4 
                 Tropomyosin alpha-4 chain 
                 55 
                 2.92 
                 1.93E-11 
                 4.04E-08 
               
               
                 LYN 
                 Tyrosine-protein kinase Lyn 
                 56 
                 1.02 
                 1.60E-06 
                 1.27E-04 
               
               
                 VASP 
                 Vasodilator-stimulated phosphoprotein 
                 57 
                 2.79 
                 3.73E-08 
                 7.11 E-06 
               
               
                 WDR1 
                 WD repeat-containing protein 1 
                 58 
                 1.57 
                 2.17E-10 
                 1.14E-07 
               
               
                 ZYX 
                 Zyxin 
                 59 
                 1.84 
                 9.71E-08 
                 1.36E-05 
               
            
           
         
       
     
     
       
         
          TABLE 2
           
               
               
               
               
               
               
               
             
               
                 Biomarkers of Group B (Significantly regulated peptides) 
               
               
                 Protein Name 
                 Uniprot Accession No. 
                 Peptide Sequence 
                 SEQ ID NO: 
                 log2 Fold-Change AD vs Control 
                 pValue 
                 Adjusted pValue 
               
             
            
               
                 Actin-related protein ⅔ complex subunit 1B 
                 015143 
                 sLESALk 
                 60 
                 2.41 
                 2.46E-10 
                 3.91E-07 
               
               
                 Alpha-enolase 
                 P06733 
                 aVEHINk 
                 61 
                 3.58 
                 1.44E-10 
                 2.71E-07 
               
               
                 Bridging integrator 2 
                 Q9UBW5 
                 aSLGTGTASPR 
                 62 
                 4.22 
                 7.71E-12 
                 6.12E-08 
               
               
                 Bridging integrator 2 
                 Q9UBW5 
                 aTASPRPSSGNIPS [p] sPTASGG G[p]sPTSPR 
                 63 
                 4.32 
                 8.25E-13 
                 1.64E-08 
               
               
                 Caldesmon 
                 Q05682 
                 gS [p] sLkIEER 
                 64 
                 5.86 
                 6.82E-12 
                 6.12E-08 
               
               
                 Caveolae-associated protein 2 
                 095810 
                 gIQNDLTk 
                 65 
                 2.73 
                 8.54E-10 
                 7.65E-07 
               
               
                 Chloride intracellular channel protein 1 
                 000299 
                 nSNPALNDNLEk 
                 66 
                 4.02 
                 8.47E-11 
                 2.24E-07 
               
               
                 Claudin-5 
                 000501 
                 rP[p]tATGDYDkk 
                 67 
                 4.94 
                 2.85E-13 
                 1.13E-08 
               
               
                 Endoplasmic reticulum chaperone BiP 
                 P11021 
                 1YGSAGPPPTGEEDTAEkDEL 
                 68 
                 3.09 
                 8.67E-10 
                 7.65E-07 
               
               
                 Filamin-A 
                 P21333 
                 aEISFEDRk 
                 69 
                 3.24 
                 8.39E-10 
                 7.65E-07 
               
               
                 Filamin-A 
                 P21333 
                 sSFTVDcSk 
                 70 
                 3.93 
                 5.28E-10 
                 5.98E-07 
               
               
                 Filamin-A 
                 P21333 
                 tPcEEILVk 
                 71 
                 3.46 
                 3.44E-10 
                 4.57E-07 
               
               
                 Junctional adhesion molecule A 
                 Q9Y624 
                 kVIYSQP[p]sAR 
                 72 
                 6.62 
                 1.88E-11 
                 1.07E-07 
               
               
                 Leucine-rich repeat flightless-interacting protein 2 
                 Q9Y608 
                 nSASATTPL [p] sGNSSR 
                 73 
                 3.07 
                 5.22E-11 
                 1.72E-07 
               
               
                 LIM and SH3 domain protein 1 
                 Q14847 
                 tQDQISNIk 
                 74 
                 2.64 
                 7.06E-10 
                 7.18E-07 
               
               
                 Linker for activation of T-cells family member 1 
                 043561 
                 [p] sQPLGGSHR 
                 75 
                 4.50 
                 1.02E-10 
                 2.39E-07 
               
               
                 Moesin 
                 P26038 
                 aQQELEEQTRR 
                 76 
                 2.34 
                 5.81E-10 
                 6.23E-07 
               
               
                 Myosin light polypeptide 6 
                 P60660 
                 s DEMNVk 
                 77 
                 3.80 
                 1.13E-11 
                 7.47E-08 
               
               
                 PDZ and LIM domain protein 1 
                 000151 
                 gcTDNLTLTVAR 
                 78 
                 2.76 
                 3.49E-10 
                 4.57E-07 
               
               
                 Platelet endothelial aggregation receptor 1 
                 Q5VY43 
                 hPP [p] sPPLR 
                 79 
                 4.21 
                 6.00E-11 
                 1.83E-07 
               
               
                 Pleckstrin 
                 P08567 
                 gSTLTSPcQDFGkR 
                 80 
                 3.17 
                 1.51E-10 
                 2.72E-07 
               
               
                 Profilin-1 
                 P07737 
                 eGVHGGLINkk 
                 81 
                 2.53 
                 4.50E-10 
                 5.25E-07 
               
               
                 Profilin-1 
                 P07737 
                 sTGGAPTFNVTVTk 
                 82 
                 2.70 
                 3.20E-10 
                 4.57E-07 
               
               
                 Protein phosphatase 1 regulatory subunit 12A 
                 O14974 
                 k[p]tGSYGALAEITASk 
                 83 
                 3.77 
                 8.10E-10 
                 7.65E-07 
               
               
                 Purine nucleoside phosphorylase 
                 P00491 
                 aNHEEVLAAGk 
                 84 
                 3.01 
                 7.94E-10 
                 7.65E-07 
               
               
                 SH3 domain-binding glutamic acid-rich-like protein 3 
                 Q9H299 
                 iQYQLVDISQDNALRDEMR 
                 85 
                 2.34 
                 6.05E-10 
                 6.31E-07 
               
               
                 SH3 domain-binding glutamic acid-rich-like protein 3 
                 Q9H299 
                 vYSTSVTGSR 
                 86 
                 2.04 
                 3.57E-10 
                 4.57E-07 
               
               
                 Src substrate cortactin 
                 Q14247 
                 akTQ[p]tPPV[p]sPAPQPTEER 
                 87 
                 3.82 
                 3.93E-12 
                 5.20E-08 
               
               
                 Talin-1 
                 Q9Y490 
                 aLDGAFTEENR 
                 88 
                 3.12 
                 4.47E-10 
                 5.25E-07 
               
               
                 Talin-1 
                 Q9Y490 
                 tIMVDDSk 
                 89 
                 2.19 
                 3.92E-10 
                 4.86E-07 
               
               
                 Transgelin-2 
                 P37802 
                 gPAYGLSR 
                 90 
                 4.44 
                 5.06E-11 
                 1.72E-07 
               
               
                 Transgelin-2 
                 P37802 
                 nMAcVQR 
                 91 
                 3.98 
                 3.40E-10 
                 4.57E-07 
               
               
                 Tropomyosin alpha-1 chain 
                 P09493 
                 aQkDEEkMEIQEIQLk 
                 92 
                 2.94 
                 1.32E-10 
                 2.61E-07 
               
               
                 Tropomyosin alpha-4 chain 
                 P67936 
                 aEFAER 
                 93 
                 4.54 
                 9.85E-11 
                 2.39E-07 
               
               
                 Tropomyosin alpha-4 chain 
                 P67936 
                 lATALQk 
                 94 
                 4.19 
                 1.12E-10 
                 2.40E-07 
               
               
                 Tropomyosin alpha-4 chain 
                 P67936 
                 aEVSELk 
                 95 
                 4.37 
                 7.28E-11 
                 2.06E-07 
               
               
                 Tropomyosin alpha-4 chain 
                 P67936 
                 ekAEGDVAALNR 
                 96 
                 4.99 
                 3.09E-10 
                 4.57E-07 
               
               
                 Tropomyosin alpha-4 chain 
                 P67936 
                 kIQALQQQADEADER 
                 97 
                 3.80 
                 2.58E-11 
                 1.25E-07 
               
               
                 Tropomyosin alpha-4 chain 
                 P67936 
                 sLEAASEk 
                 98 
                 5.18 
                 2.82E-11 
                 1.25E-07 
               
               
                 Tropomyosin alpha-4 chain 
                 P67936 
                 tIDDLEEk 
                 99 
                 5.75 
                 4.88E-11 
                 1.72E-07 
               
               
                 UV excision repair protein RAD23 homolog B 
                 P54727 
                 nQPQFQQMR 
                 100 
                 2.76 
                 2.02E-10 
                 3.49E-07 
               
               
                 WD repeat-containing protein 1 
                 075083 
                 lYSILGTTLkDEGk 
                 101 
                 2.08 
                 2.27E-10 
                 3.75E-07 
               
               
                 WD repeat-containing protein 1 
                 O75083 
                 vINSVDIk 
                 102 
                 2.12 
                 8.08E-10 
                 7.65E-07 
               
               
                 WD repeat-containing protein 1 
                 075083 
                 yEYQPFAGk 
                 103 
                 2.25 
                 1.15E-10 
                 2.40E-07 
               
               
                 Zyxin 
                 Q15942 
                 gPPASSPAPAPkF[p]sPVTPk 
                 104 
                 3.78 
                 5.70E-10 
                 6.23E-07 
               
            
           
         
       
     
     
       
         
          TABLE 3
           
               
               
               
               
               
               
               
             
               
                 Biomarkers of Group C (Additional Phosphopeptides) 
               
               
                 Protein Name 
                 Uniprot Accession No. 
                 Peptide Sequence 
                 SEQ ID NO: 
                 log2 Fold-Change AD vs Control 
                 pValue 
                 Adjusted pValue 
               
             
            
               
                 Claudin-5 
                 O00501 
                 rP [p ]tATGDYDkk 
                 105 
                 4.94 
                 3.07E-13 
                 2.87E-09 
               
               
                 Protein phosphatase 1 regulatory subunit 12A 
                 O14974 
                 k [p] tGSYGALAEITASk 
                 106 
                 3.77 
                 4.37E-10 
                 3.75E-07 
               
               
                 Linker for activation of T-cells family member 1 
                 043561 
                 [p] sPQPLGGSHR 
                 107 
                 4.50 
                 5.19E-11 
                 6.92E-08 
               
               
                 Caveolae-associated protein 2 
                 095810 
                 kVD [p] sLkk 
                 108 
                 6.10 
                 4.26E-09 
                 1.99E-06 
               
               
                 Platelet glycoprotein Ib alpha chain 
                 P07359 
                 ySGH [p] sL 
                 109 
                 6.42 
                 6.24E-10 
                 4.16E-07 
               
               
                 Platelet glycoprotein Ib beta chain 
                 P13224 
                 l [p] sLTDPLVAER 
                 110 
                 4.70 
                 1.00E-09 
                 6.24E-07 
               
               
                 Filamin-A 
                 P21333; 075369 
                 vkE [p] sITR 
                 111 
                 5.92 
                 1.57E-09 
                 8.76E-07 
               
               
                 Transgelin-2 
                 P37802 
                 nF [p] sDNQLQEGk 
                 112 
                 2.44 
                 1.59E-09 
                 8.76E-07 
               
               
                 Caldesmon 
                 Q05682 
                 gS [p] sLkIEER 
                 113 
                 5.86 
                 3.16E-12 
                 9.85E-09 
               
               
                 Nexilin 
                 Q0ZGT2 
                 eMLA [p] sDDEEDVSSkVEk 
                 114 
                 4.98 
                 4.42E-10 
                 3.75E-07 
               
               
                 Src substrate cortactin 
                 Q14247 
                 akTQ [p] tPPV [p] sPAPQPTEER 
                 115 
                 3.82 
                 5.39E-12 
                 1.15E-08 
               
               
                 Ras GTPase-activating protein 3 
                 Q14644 
                 yG [p] sQEHPIGDk 
                 116 
                 4.07 
                 4.25E-09 
                 1.99E-06 
               
               
                 Eukaryotic translation initiation factor 4H 
                 Q15056 
                 aYSSFGGGRG [p] sR 
                 117 
                 3.05 
                 1.73E-09 
                 8.95E-07 
               
               
                 Lethal(2) giant larvae protein homolog 1 
                 Q15334 
                 iRE [p] sPk 
                 118 
                 2.78 
                 4.93E-09 
                 2.19E-06 
               
               
                 Zyxin 
                 Q15942 
                 gPPASSPAPAPkF [p] sPVTPk 
                 119 
                 3.78 
                 3.39E-10 
                 3.51E-07 
               
               
                 Zyxin 
                 Q15942 
                 [p] sPGAPGPLTLk 
                 120 
                 3.96 
                 5.43E-10 
                 3.90E-07 
               
               
                 Platelet endothelial aggregation receptor 1 
                 Q5VY43 
                 hPP [p] sPPLR 
                 121 
                 4.21 
                 3.75E-11 
                 5.83E-08 
               
               
                 Synaptotagmin-like protein 4 
                 Q96C24 
                 rD [p] sLDkSGLFPEWk 
                 122 
                 4.36 
                 5.21E-10 
                 3.90E-07 
               
               
                 Bridging integrator 2 
                 Q9UBW5 
                 aTASPRPSSGNIPS [p] sPTASGG G [p] sPTSPR 
                 123 
                 4.32 
                 8.37E-13 
                 3.91E-09 
               
               
                 Leucine-rich repeat flightless-interacting protein 2 
                 Q9Y608 
                 nSASATTPL [p] sGNSSR 
                 124 
                 3.07 
                 8.07E-11 
                 9.41E-08 
               
               
                 Junctional adhesion molecule A 
                 Q9Y624 
                 kVIYSQP [p] sAR 
                 125 
                 6.62 
                 6.15E-12 
                 1.15E-08 
               
            
           
         
       
     
     
       
         
          TABLE 4A
           
               
               
               
               
               
               
               
               
             
               
                 Filamin A peptides detected by TMTcalibrator™ mass spectrometry 
               
               
                 FLNA Peptide 
                 SEQ ID NO: 
                 Relative Expression Controls 
               
               
                 Old 
                 Old 
                 Young 
                 Young 
                 Young 
                 Old 
               
             
            
               
                 aEAGVPAEFSIWTR 
                 126 
                 -0.58 
                 -0.57 
                 -0.27 
                 0.28 
                 -0.59 
                 -0.50 
               
               
                 aEGPGLSR 
                 127 
                 -0.93 
                 -1.48 
                 -1.08 
                 -1.18 
                 -0.89 
                 -1.34 
               
               
                 vkE[p]sITR 
                 128 
                 -0.66 
                 0.42 
                 0.28 
                 -0.22 
                 0.87 
                 -3.28 
               
               
                 aEISFEDRk 
                 129 
                 -1.54 
                 -1.29 
                 -1.79 
                 -1.11 
                 -1.46 
                 -1.52 
               
               
                 aFGPGLQGGSAGSPAR 
                 130 
                 -0.15 
                 -0.46 
                 0.06 
                 0.32 
                 -0.30 
                 -0.59 
               
               
                 aGGPGLER 
                 131 
                 -1.77 
                 -1.17 
                 -1.97 
                 -2.91 
                 -1.92 
                 -0.70 
               
               
                 aGQSAAGAAPGGGVDTR 
                 132 
                 0.37 
                 1.10 
                 ND 
                 0.39 
                 0.46 
                 1.25 
               
               
                 aGVAPLQVk 
                 133 
                 -0.62 
                 -0.53 
                 0.13 
                 0.07 
                 -0.31 
                 -0.38 
               
               
                 aNLPQSFQVDTSk 
                 134 
                 0.76 
                 0.23 
                 0.80 
                 1.40 
                 1.16 
                 1.07 
               
               
                 aSGPGLNTTGVPASLPVEFTIDAk 
                 135 
                 2.85 
                 2.44 
                 3.06 
                 2.86 
                 2.55 
                 2.53 
               
               
                 aTcAPQHGAPGPGPADASk 
                 136 
                 -4.90 
                 -4.73 
                 -7.14 
                 -2.99 
                 -2.65 
                 -3.04 
               
               
                 aWGPGLEGGVVGk 
                 137 
                 0.68 
                 0.41 
                 0.17 
                 0.80 
                 0.54 
                 0.42 
               
               
                 aP[p]sVANVGSHcDLSLk 
                 138 
                 ND 
                 ND 
                 ND 
                 -2.66 
                 ND 
                 ND 
               
               
                 aYGPGIEPTGNMVk 
                 139 
                 -1.04 
                 -0.98 
                 -0.31 
                 0.05 
                 -1.03 
                 -1.13 
               
               
                 cSGPGLER 
                 140 
                 -3.00 
                 -2.90 
                 -2.04 
                 -2.35 
                 -1.86 
                 -1.60 
               
               
                 cSGPGLSPGMVR 
                 141 
                 -1.15 
                 -1.61 
                 -0.43 
                 0.03 
                 -0.66 
                 -0.13 
               
               
                 dAGEGGLSLAIEGPSk 
                 142 
                 -0.87 
                 -1.66 
                 -1.37 
                 -0.56 
                 -0.29 
                 -0.98 
               
               
                 dAGEGLLAVQITDPEGkPk 
                 143 
                 -0.43 
                 -0.47 
                 -0.67 
                 -0.28 
                 -0.17 
                 -0.41 
               
               
                 dAPQDFHPDR 
                 144 
                 -0.41 
                 ND 
                 ND 
                 ND 
                 -0.59 
                 -1.03 
               
               
                 cSGPGL[p]sPGMVR 
                 145 
                 -2.43 
                 -2.29 
                 -2.90 
                 -2.80 
                 -2.93 
                 -0.76 
               
               
                 dGScGVAYVVQEPGDYEVSVk 
                 146 
                 1.17 
                 1.02 
                 0.62 
                 0.97 
                 0.75 
                 0.59 
               
               
                 dLAEDAPWk 
                 147 
                 0.01 
                 -0.02 
                 -0.16 
                 0.53 
                 -0.19 
                 -0.06 
               
               
                 dVDIIDHHDNTYTVk 
                 148 
                 -3.11 
                 -2.85 
                 ND 
                 -3.37 
                 -2.67 
                 -3.38 
               
               
                 eAGAGGLAIAVEGPSk 
                 149 
                 0.81 
                 0.45 
                 0.75 
                 1.04 
                 1.10 
                 0.74 
               
               
                 eEPcLLkR 
                 150 
                 -1.73 
                 -0.81 
                 -0.68 
                 -0.67 
                 -0.74 
                 -0.93 
               
               
                 eGPYSISVLYGDEEVPR 
                 151 
                 ND 
                 1.12 
                 1.50 
                 1.35 
                 0.94 
                 ND 
               
               
                 eNGVYLIDVk 
                 152 
                 -1.03 
                 -1.04 
                 -1.18 
                 -0.29 
                 -0.81 
                 -1.12 
               
               
                 eTGEHLVHVk 
                 153 
                 -2.33 
                 -1.72 
                 -1.86 
                 -1.78 
                 -1.28 
                 -1.46 
               
               
                 fNEEHIPDSPFVVPVASPSGDAR 
                 154 
                 0.07 
                 0.01 
                 0.42 
                 0.54 
                 -0.22 
                 0.24 
               
               
                 fTVETR 
                 155 
                 -1.10 
                 -0.99 
                 -0.80 
                 -0.60 
                 -0.63 
                 -0.57 
               
               
                 gAGTGGLGLAVPSEAk 
                 156 
                 1.78 
                 1.80 
                 1.59 
                 1.93 
                 1.86 
                 1.51 
               
               
                 gLVEPVDVVDNADGTQTVNYVPSR 
                 157 
                 0.02 
                 -0.49 
                 -2.34 
                 0.62 
                 0.75 
                 -2.43 
               
               
                 gTVEPQLEAR 
                 158 
                 -0.46 
                 0.32 
                 0.22 
                 -0.12 
                 -0.01 
                 -0.38 
               
               
                 iEcDDkGDGScDVR 
                 159 
                 -2.11 
                 -2.80 
                 -2.80 
                 -1.44 
                 -1.51 
                 -1.90 
               
               
                 iPEISIQDMTAQV[?p]t[?p]sPSGk 
                 160 
                 0.75 
                 0.45 
                 0.14 
                 1.07 
                 0.74 
                 0.93 
               
               
                 iVGPSGAAVPck 
                 161 
                 2.04 
                 1.46 
                 0.94 
                 2.22 
                 1.66 
                 1.88 
               
               
                 kGEITGEVR 
                 162 
                 -1.69 
                 -1.31 
                 -1.34 
                 -0.80 
                 -1.21 
                 -1.42 
               
               
                 lQVEPAVDTGVQcYGPGIEGQGVFR 
                 163 
                 -0.56 
                 ND 
                 -0.62 
                 0.18 
                 0.38 
                 -0.74 
               
               
                 mDcQEcPEGYR 
                 164 
                 ND 
                 ND 
                 0.82 
                 -0.29 
                 1.31 
                 ND 
               
               
                 mScMDNk 
                 165 
                 -0.83 
                 -0.20 
                 0.50 
                 -0.29 
                 -0.13 
                 -1.06 
               
               
                 rAP[p]sVANVGSHcDLSLk 
                 166 
                 ND 
                 -1.99 
                 -0.99 
                 -0.48 
                 -1.63 
                 -2.08 
               
               
                 sAGQGEVLVYVEDPAGHQEEAk 
                 167 
                 2.23 
                 2.21 
                 2.75 
                 0.73 
                 1.88 
                 1.96 
               
               
                 sPFEVYVDk 
                 168 
                 -0.28 
                 -0.24 
                 0.11 
                 -0.30 
                 -0.48 
                 -0.22 
               
               
                 sPFSVAVSPSLDLSk 
                 169 
                 2.28 
                 2.21 
                 2.23 
                 2.47 
                 2.08 
                 2.12 
               
               
                 sPYTVTVGQAcNPSAcR 
                 170 
                 ND 
                 ND 
                 1.47 
                 ND 
                 ND 
                 ND 
               
               
                 sSFTVDcSk 
                 171 
                 -1.41 
                 -1.36 
                 -1.85 
                 -0.52 
                 -0.79 
                 -0.75 
               
               
                 tFSVWYVPEVTGTHk 
                 172 
                 0.12 
                 0.06 
                 0.41 
                 0.48 
                 0.17 
                 0.62 
               
               
                 tGVAVNkPAEFTVDAk 
                 173 
                 0.11 
                 -0.52 
                 0.12 
                 0.32 
                 0.08 
                 0.78 
               
               
                 tHEAEIVEGENHTYcIR 
                 174 
                 ND 
                 ND 
                 ND 
                 ND 
                 2.33 
                 ND 
               
               
                 tPcEEILVk 
                 175 
                 -0.87 
                 -0.84 
                 -0.62 
                 -0.61 
                 -0.78 
                 -1.03 
               
               
                 vANPSGNLTETYVQDR 
                 176 
                 -0.55 
                 -0.20 
                 -0.99 
                 -0.98 
                 -0.35 
                 -0.24 
               
               
                 vAQPTITDNkDGTVTVR 
                 177 
                 -2.22 
                 -1.89 
                 -2.24 
                 -1.76 
                 -1.71 
                 -1.60 
               
               
                 vDVGkDQEFTVk 
                 178 
                 -0.66 
                 -0.67 
                 -1.21 
                 -0.60 
                 -1.04 
                 -0.76 
               
               
                 vEPGLGADNSVVR 
                 179 
                 0.44 
                 0.23 
                 0.36 
                 1.01 
                 0.63 
                 0.14 
               
               
                 vQVQDNEGcPVEALVk 
                 180 
                 0.01 
                 0.03 
                 0.03 
                 -0.39 
                 0.13 
                 -0.05 
               
               
                 vTAQGPGLEPSGNIANk 
                 181 
                 -1.13 
                 -1.81 
                 -1.65 
                 -0.76 
                 -0.75 
                 -1.04 
               
               
                 vTVLFAGQHIAk 
                 182 
                 2.53 
                 2.33 
                 2.78 
                 3.23 
                 2.63 
                 2.48 
               
               
                 wGDEHIPGSPYR 
                 183 
                 -0.27 
                 -0.87 
                 -0.82 
                 -0.67 
                 -0.51 
                 -0.24 
               
               
                 yGGDEIPFSPYR 
                 184 
                 -0.60 
                 -0.55 
                 -1.45 
                 -0.44 
                 -0.70 
                 -0.90 
               
               
                 yTPVQQGPVGVNVTYGGDPIPk 
                 185 
                 -0.04 
                 -0.21 
                 -0.04 
                 0.27 
                 -0.74 
                 -0.02 
               
            
           
         
       
     
     
       
         
          TABLE 4B
           
               
               
               
               
               
               
               
               
             
               
                 Filamin A peptides detected by TMTcalibrator™ mass spectrometry 
               
               
                 FLNA Peptide 
                 SEQ ID NO: 
                 Relative Expression Alzheimer’s Disease Subjects 
               
             
            
               
                 aEAGVPAEFSIWTR 
                 126 
                 1.66 
                 1.24 
                 0.88 
                 0.66 
                 1.93 
                 1.58 
               
               
                 aEGPGLSR 
                 127 
                 1.74 
                 1.46 
                 1.36 
                 0.85 
                 2.02 
                 1.49 
               
               
                 vkE[p]sITR 
                 128 
                 6.01 
                 5.42 
                 5.24 
                 4.89 
                 6.08 
                 5.92 
               
               
                 aEISFEDRk 
                 129 
                 2.07 
                 1.72 
                 1.46 
                 0.99 
                 2.19 
                 2.01 
               
               
                 aFGPGLQGGSAGSPAR 
                 130 
                 2.41 
                 1.94 
                 1.86 
                 1.44 
                 2.67 
                 2.43 
               
               
                 aGGPGLER 
                 131 
                 1.54 
                 1.17 
                 0.73 
                 0.40 
                 0.36 
                 1.10 
               
               
                 aGQSAAGAAPGGGVDTR 
                 132 
                 1.64 
                 1.81 
                 0.93 
                 0.88 
                 1.71 
                 0.86 
               
               
                 aGVAPLQVk 
                 133 
                 0.97 
                 0.79 
                 0.68 
                 0.67 
                 0.79 
                 0.79 
               
               
                 aNLPQSFQVDTSk 
                 134 
                 2.20 
                 2.03 
                 1.76 
                 1.76 
                 2.75 
                 2.11 
               
               
                 aSGPGLNTTGVPASLPVEFTIDAk 
                 135 
                 2.56 
                 2.63 
                 2.61 
                 2.70 
                 2.80 
                 2.70 
               
               
                 aTcAPQHGAPGPGPADASk 
                 136 
                 1.47 
                 1.43 
                 0.30 
                 0.24 
                 1.52 
                 0.99 
               
               
                 aWGPGLEGGVVGk 
                 137 
                 2.00 
                 1.48 
                 1.36 
                 1.18 
                 2.14 
                 1.73 
               
               
                 aP[p]sVANVGSHcDLSLk 
                 138 
                 -0.44 
                 -1.63 
                 -1.40 
                 -1.67 
                 -0.85 
                 -0.39 
               
               
                 aYGPGIEPTGNMVk 
                 139 
                 1.42 
                 0.83 
                 0.89 
                 0.23 
                 1.67 
                 1.21 
               
               
                 cSGPGLER 
                 140 
                 -2.26 
                 -2.19 
                 -3.33 
                 -1.89 
                 -1.03 
                 -2.66 
               
               
                 cSGPGLSPGMVR 
                 141 
                 -1.38 
                 -0.49 
                 -0.60 
                 -0.53 
                 -0.45 
                 -1.23 
               
               
                 dAGEGGLSLAIEGPSk 
                 142 
                 1.34 
                 0.94 
                 1.06 
                 0.31 
                 1.14 
                 1.28 
               
               
                 dAGEGLLAVQITDPEGkPk 
                 143 
                 1.00 
                 0.99 
                 0.82 
                 0.42 
                 1.11 
                 1.18 
               
               
                 dAPQDFHPDR 
                 144 
                 1.68 
                 1.12 
                 1.52 
                 0.95 
                 2.50 
                 1.78 
               
               
                 cSGPGL[p]sPGMVR 
                 145 
                 -2.00 
                 -2.06 
                 -3.65 
                 -1.56 
                 -0.20 
                 -1.48 
               
               
                 dGScGVAYVVQEPGDYEVSVk 
                 146 
                 1.73 
                 1.44 
                 1.46 
                 1.37 
                 1.09 
                 1.11 
               
               
                 dLAEDAPWk 
                 147 
                 0.87 
                 0.36 
                 0.52 
                 0.56 
                 0.86 
                 0.52 
               
               
                 dVDIIDHHDNTYTVk 
                 148 
                 1.81 
                 1.30 
                 0.77 
                 0.52 
                 2.17 
                 1.61 
               
               
                 eAGAGGLAIAVEGPSk 
                 149 
                 2.40 
                 1.86 
                 2.02 
                 1.70 
                 2.42 
                 2.35 
               
               
                 eEPcLLkR 
                 150 
                 1.08 
                 0.77 
                 1.28 
                 0.57 
                 2.04 
                 0.93 
               
               
                 eGPYSISVLYGDEEVPR 
                 151 
                 1.32 
                 1.47 
                 1.39 
                 0.82 
                 1.40 
                 0.80 
               
               
                 eNGVYLIDVk 
                 152 
                 0.47 
                 0.49 
                 0.04 
                 -0.05 
                 0.11 
                 0.31 
               
               
                 eTGEHLVHVk 
                 153 
                 2.43 
                 1.83 
                 2.04 
                 1.31 
                 2.65 
                 2.53 
               
               
                 fNEEHIPDSPFVVPVASPSGDAR 
                 154 
                 1.00 
                 0.97 
                 0.87 
                 0.62 
                 1.60 
                 1.20 
               
               
                 fTVETR 
                 155 
                 0.68 
                 0.38 
                 0.22 
                 0.03 
                 0.87 
                 0.69 
               
               
                 gAGTGGLGLAVEGPSEAk 
                 156 
                 3.10 
                 2.81 
                 2.97 
                 2.52 
                 3.11 
                 2.99 
               
               
                 gLVEPVDVVDNADGTQTVNYVPSR 
                 157 
                 -2.55 
                 -2.60 
                 -3.00 
                 -2.52 
                 -1.59 
                 0.29 
               
               
                 gTVEPQLEAR 
                 158 
                 1.23 
                 0.99 
                 0.73 
                 0.31 
                 1.73 
                 1.45 
               
               
                 iEcDDkGDGScDVR 
                 159 
                 2.15 
                 1.62 
                 1.26 
                 0.89 
                 2.50 
                 1.64 
               
               
                 iPEISIQDMTAQV[?p]t[?p]sPSGk 
                 160 
                 2.97 
                 1.33 
                 2.81 
                 1.61 
                 1.48 
                 3.83 
               
               
                 iVGPSGAAVPck 
                 161 
                 2.32 
                 2.34 
                 1.99 
                 2.37 
                 2.66 
                 2.20 
               
               
                 kGEITGEVR 
                 162 
                 1.05 
                 1.01 
                 0.99 
                 0.38 
                 0.58 
                 0.72 
               
               
                 lQVEPAVDTSGVQcYGPGIEGQGVFR 
                 163 
                 0.78 
                 0.38 
                 1.00 
                 0.71 
                 0.91 
                 0.89 
               
               
                 mDcQEcPEGYR 
                 164 
                 1.54 
                 0.84 
                 1.20 
                 0.67 
                 1.96 
                 -2.59 
               
               
                 mScMDNk 
                 165 
                 3.14 
                 2.47 
                 2.43 
                 1.67 
                 2.65 
                 2.50 
               
               
                 rAP[p]sVANVGSHcDLSLk 
                 166 
                 -0.96 
                 -1.80 
                 -1.80 
                 -0.55 
                 -1.04 
                 -0.68 
               
               
                 sAGQGEVLVYVEDPAGHQEEAk 
                 167 
                 1.52 
                 1.08 
                 0.80 
                 1.01 
                 1.96 
                 1.57 
               
               
                 sPFEVYVDk 
                 168 
                 0.75 
                 0.30 
                 0.27 
                 0.12 
                 1.09 
                 0.48 
               
               
                 sPFSVAVSPSLDLSk 
                 169 
                 2.42 
                 2.17 
                 2.45 
                 2.38 
                 2.58 
                 2.52 
               
               
                 sPYTVTVGQAcNPSAcR 
                 170 
                 2.35 
                 2.13 
                 2.04 
                 1.84 
                 2.33 
                 2.73 
               
               
                 sSFTVDcSk 
                 171 
                 3.08 
                 2.50 
                 2.83 
                 1.77 
                 3.19 
                 3.18 
               
               
                 tFSVWYVPEVTGTHk 
                 172 
                 0.61 
                 0.49 
                 0.66 
                 0.67 
                 1.24 
                 0.73 
               
               
                 tGVAVNkPAEFTVDAk 
                 173 
                 1.87 
                 1.52 
                 1.35 
                 1.15 
                 1.02 
                 1.47 
               
               
                 tHEAEIVEGENHTYcIR 
                 174 
                 0.94 
                 1.61 
                 1.72 
                 2.26 
                 2.14 
                 1.34 
               
               
                 tPcEEILVk 
                 175 
                 2.77 
                 2.22 
                 2.63 
                 1.56 
                 3.01 
                 3.02 
               
               
                 vANPSGNLTETYVQDR 
                 176 
                 2.01 
                 1.66 
                 1.36 
                 0.96 
                 2.01 
                 1.87 
               
               
                 vAQPTITDNkDGTVTVR 
                 177 
                 0.38 
                 0.06 
                 0.29 
                 -0.40 
                 0.55 
                 -0.03 
               
               
                 vDVGkDQEFTVk 
                 178 
                 1.74 
                 1.40 
                 1.28 
                 0.35 
                 2.16 
                 1.72 
               
               
                 vEPGLGADNSVVR 
                 179 
                 2.42 
                 1.92 
                 1.93 
                 1.40 
                 1.92 
                 2.14 
               
               
                 vQVQDNEGcPVEALVk 
                 180 
                 1.74 
                 1.30 
                 1.31 
                 0.58 
                 2.00 
                 1.52 
               
               
                 vTAQGPGLEPSGNIANk 
                 181 
                 1.37 
                 1.11 
                 0.93 
                 0.66 
                 1.88 
                 1.49 
               
               
                 vTVLFAGQHIAk 
                 182 
                 3.10 
                 3.11 
                 2.85 
                 2.93 
                 3.28 
                 3.37 
               
               
                 wGDEHIPGSPYR 
                 183 
                 1.19 
                 1.04 
                 1.13 
                 0.30 
                 0.61 
                 1.38 
               
               
                 yGGDEIPFSPYR 
                 184 
                 0.50 
                 0.32 
                 0.21 
                 -0.04 
                 0.18 
                 0.57 
               
               
                 yTPVQQGPVGVNVTYGGDPIPk 
                 185 
                 1.76 
                 1.42 
                 1.45 
                 1.00 
                 2.04 
                 1.63 
               
            
           
         
       
     
     EXAMPLES 
     It should be understood that the above-described embodiments and the following examples are given by way of illustration, not limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the present description. 
     Example 1: TMTcalibrator™ Analysis of Histopaque®-1077 Prepared Alzheimer’s Plasma and EDTA Control Plasma 
     Samples 
     Human plasma samples from 6 controls (3 elderly controls, 3 young cognitive intact) were drawn into Vacutainer® tubes (Becton, Dickinson and Company Franklin Lakes, NJ) containing K2EDTA. [Plasma from controls were negative (-) for the 90 kDa FLNA biomarker as revealed by Western blot using a phospho-specific rabbit polyclonal antibody specific for pS2152 (Origene TA313881, Origene Technologies Rockville, MD).] Within 30 minutes after being drawn, the blood was centrifuged blood at approximately 1000 X G for 15 min, preferably at 4-5° C. Within 30 min of centrifuging, plasma was collected. Control plasma was then combined with 5% volume/volume of 20X protease and phosphatase inhibitor cocktails and added, thoroughly mixed by vortexing for 1 minute and aliquoted. The protease-phosphate inhibitor cocktail was prepared by dissolving 1X Roche PhosStop EASYpak together with 1X Roche complete tablets mini EDTA free EASYpack (Thomas Scientific, Swedesboro, NJ) into 500 ml of distilled water. Human plasma samples from 6 AD patients were drawn into Vacutainer® tubes and transported to the laboratory for processing. Human plasma from AD patients were positive (+) for the 90 kDa FLNA biomarker as revealed by Western blot using a phospho-specific rabbit polyclonal antibody specific for pS2152 (Origene TA313881, Origene Technologies Rockville, MD). At the laboratory, 4 ml of the whole blood from the Vacutainer® tube were layered onto 4 ml of Histopaque®-1077 (Sigma-Aldrich) in a 14 ml disposable tube. The disposable tube was centrifuged at 400 g for 30 min at room temperature after which plasma was transferred to 1.5 ml Eppendorf tubes for storage and 5% volume/volume of 20X protease and phosphatase inhibitor cocktails and added, thoroughly mixed by vortexing for 1 minute and aliquoted for use in biomarker assays. Aliquots were stored at -80 C. An aliquot of three post-mortem AD brain (Braak Stage IV-VI) lysates was provided by Proteome Sciences and used as a trigger sample of brain-derived proteins. 
     Analytical Method 
     For this experiment, a TMTcalibrator™ phosphoproteomic analysis ( FIG.  1   ) was performed using 12 AD plasma samples with AD brain tissue used as a trigger sample. Plasma samples were depleted from high-abundant proteins using Top14 Abundant Protein Depletion spin columns. Proteins were digested using trypsin and the peptides labelled with TMTpro™ and mixed to generate one TMTpro™ 16-plex sample (reagents available from Thermo Fisher Scientific, Waltham, MA, USA), Four TMTproTM channels were used for the AD brain tissue trigger. 
     The TMTpro™ 16-plex sample was split into an aliquot for total proteome analysis (non-enriched) and an aliquot for phosphoproteome analysis (phosphopeptide enriched). After phosphopeptide enrichment, 6 phosphopeptide-enriched and 6 non-enriched fractions were generated. Each fraction was subjected to LC-MS2 analysis using a high-performance Orbitrap Fusion™ Tribrid™ mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA. ) using a data-dependent acquisition method (using an inclusion list for FLNA peptides for the non-enriched fractions). Raw data were searched using Proteome Discoverer™ v2.5 (Thermo Fisher Scientific). Data were further processed using a proprietary bioinformatics pipeline involving filtering, normalisation, biostatistics, annotation and functional analysis. Box-plots of the proteins of interest were also generated. 
     Sample Management 
     Upon receipt, all samples were visually inspected to assess the absence of thawing, correct labelling and general integrity. Samples were stored at -80° C. Details were entered into the Laboratory Information Management System (LIMS) under a unique reference number. 
     Brain Lysis 
     Available protein from pooled AD brain tissue lysates was used as a calibrator/trigger brain sample. 
     Protein Concentration Measurement and SDS-PAGE 
     The protein concentration of plasma, depleted plasma and brain lysate samples were determined by Bradford protein assay and each sample visualised by Coomassie stained (Imperial Stain, Pierce, Thermo Fisher Scientific) SDS-PAGE 4-20% gradient gels (Criterion, Biorad). 
     Protein Depletion 
     35 µL of plasma were depleted using HighSelect Top14 Abundant Protein Depletion (Pierce, Thermo Fisher Scientific) based on the manufacturer’s protocol. For several samples multipe aliquots of 35 µl each were depleted to obtain sufficient amounts (Table S2). 
     Digestion and TMTpro Labelling 
     Calibrator/Trigger brain sample: 6.6 mg of the pooled brain lysate was reduced (dithiothreitol), alkylated (iodoacetamide), digested (trypsin) to generate peptides, desalted (SepPak® tC18 cartridges, Waters Corp., Milford, MA, USA), aliquoted into 4 portions each reflecting a 1:4:6:10 ratio, and lyophilised to dryness. 
     Analytical depleted plasma samples: 180 µg per individual depleted plasma sample was used. Depleted plasma samples were brought to equal volumes. Samples were reduced, alkylated and digested with trypsin to generate peptides, and after desalting (SepPak ®  tC18 cartridges) samples were lyophilised to dryness. 
     Dry peptides (from depleted plasma and brain samples) were dissolved in TEAB/ACN buffer. Peptides were mixed with their respective TMTpro™ reagent (labelling plan shown in Table 2). TMTpro™ labelled samples were treated with hydroxylamine and the labelled digests were pooled to generate the TMTpro™ 16-plex sample containing 12 depleted plasma analytical samples (12 × 180 µg = 2160 µg) + pooled brain digest mixture (about 3780 µg in 4 channels with a ratio of 1:4:6:10) in a protein mass ratio of 1:1.75). 50 µg of the mixtures were purified by solid phase extractions to be used for assessment of labelling efficiency and reporter ion distributions (equimolarity check). Two portions each of 100 µg were taken off as samples for basic-reversed fractionation for the non-enriched arm (including a backup sample) and about 5,690 µg portions for phosphopeptide enrichment. 
     Phosphopeptide Enrichment 
     About 5,690 µg of the TMTpro™ 16-plex sample was enriched for phosphopeptides over two columns of the High Select™ Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scientific Pierce, cat. no. A32992) according to manufacturer’s instructions and combined yielding one pooled phosphopeptide sample. 
     Basic Reversed-Phase Fractionation (bRP) 
     bRP fractionation was conducted for the TMTpro™ 16plex taking A) 100 µg of non-enriched samples and B) enriched phosphopeptides. Here, the Thermo Scientific™ Pierce™ High pH Reversed-Phase Peptide Fractionation Kit (cat. no. 84868) was used according to manufacturer’s instructions. 
     Liquid Chromatography Mass Spectrometry (LC-MS/MS) 
     Each of the individual fractions (6 phosphopeptide enriched and 6 non-enriched fractions per 16plex) was analysed by LC-MS/MS using an EASYnLC-1000 system coupled to an Orbitrap FusionTM TribridTM Mass Spectrometer (both Thermo Scientific). Peptides were resuspended in 2% acetonitrile (ACN) with 0.1% formic acid (FA). From the thermostatted autosampler, samples (about5% of non-enriched and about 25% of phosphopeptide enriched fractions) were loaded onto a 2 cm 75 µm inner diameter (ID) C18 Acclaim PepMap 100 trapping column (Thermo Scientific, PN 164946) and resolved using an increasing gradient of ACN in 0.1% FA through a 50 cm 75 µm ID EasySpray analytical column (Thermo Scientific, PN ES803A) at a flow rate of 200 nL/min. Gradient starting conditions were 8% ACN for non-enriched fractions and 10% ACN for phospho-enriched fractions. The percentage of organic solvent was continuously increased to a maximum of 30 % ACN. 
     Peptide mass spectra were acquired throughout the entire chromatographic run (180 minutes). The mass spectrometer was operated in data dependent mode with full scans at 120.000 resolution and MS2 fragment scans acquired at 50.000 resolution. The duty cycle was set to 3 seconds, meaning a full scan was acquired at least every 3 seconds, followed by MS2 scans of fragmented precursors picked from the most abundant targets. After being fragmented once, precursors were excluded from additional fragmentation for 30 seconds. 
     Computational Mass Spectrometry 
     In total, 12 separate raw mass spectrometry data files (12 fractions - 6 enriched &amp; 6 non-enriched) were searched in Proteome Discoverer (PD) v2.5 (Thermo Scientific) using the SEQUEST HT Search algorithm. The raw spectra were searched against a uniprot reviewed human database (version January 2021), with full tryptic specificity and up to two missed cleavages. All spectra that were not matched to a peptide with high confidence, were searched again with semi-tryptic specificity to account for potential cleavage products. Spectra still unmatched after the semi-tryptic search, were searched again with no cleavage specificity. TMTpro™ modification of N-termini and lysine residues, as well as carbamidomethylation of cysteine, were set as static modifications; oxidation of methionine was considered as a variable modification. Phosphorylation in serine, threonine and tyrosine residues was set as variable modification in the searches of phosphopeptide enriched fractions only. 
     The precursor mass tolerance was set to 20 ppm, while the fragment mass tolerance was set to 0.02 Da. The false discovery rate was controlled at 1% on PSM level by the Percolator node incorporated in Proteome Discoverer. The reporter ions quantifier node was set up to extract the raw intensity values for TMTpro™ 16plex mono-isotopic ions (126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, 131N, 131C, 132N, 132C, 133N, 133C, 134N). All raw reporter ion intensity values were exported to tab delimited text files for further processing and bioinformatic analysis. 
     Bioinfonnatics Data Analysis 
     Statistical analysis was conducted using internally developed software written in R statistical programming language (R Core Team 2017). All data integration tools were developed to work with TMT® labelled MS data and included functionality for dealing with isolation interference [Savitski, M., et al. “Measuring and managing ratio compression for accurate iTRAQ/TMT quantification.”  J. Proteome Research  12.8 (2013): 3586-3598], isotopic crosstalk [Rauniyar, N., et al. “Isobaric labeling-based relative quantification in shotgun proteomics.”  J. Proteome Research  13.12 (2014): 5293-5309], PSM normalisation, and summarisation into peptides. 
     Statistical Analysis 
     Statistical analysis was conducted using in-house software and was based on Linear Modelling. [T., Tibshirani, et al., The Elements of Statistical Learning: Data Mining, Inference, and Prediction (Vol. 2, pp. 1-758). New York: Springer (2009).] For feature selection moderated t-statistics were selected (Ritchie, M., et al. “Limma Powers Differential Expression Analyses for RNA-sequencing and Microarray Studies.”  Nucleic Acids Research  43.7 (2015): e47-e47; Phipson, B., et al. “Robust Hyperparameter Estimation Protects Against Hypervariable Genes and Improves Power to Detect Differential Expression.”  Annals of Applied Statistics  10.2 (2016): 946.)], which effectively borrows information from the ensemble of features to aid with inference about each individual feature and is useful for small data sets. 
     Analysis was performed for 3 different contrasts: Contrast 1 = AD vs Control (old); Contrast 2 = AD vs Control (young); and Contrast 3 = AD vs Control (all). For each contrast, the fold changes, p-values and adjusted p-values (calculated using the Benjamini-Hochberg FDR) are provided. The LIMMA p-values are based on the moderated t-statistics (Ritchie, Phipson) and are the result of information borrowing across all features to overcome the problem of small sample sizes. Multiple testing corrections were applied using the Benjamini-Hochberg procedure. 
     Sample Assessment 
     Protein determination by modified Bradford assay showed that protein concentrations of plasma samples were between 41.9 and 73.6 µg/µl (mean: about 59 µg/µl). AD samples showed a lower mean protein concentration versus controls (48.7 vs 68.5 µg/µl), SDS-PAGE of all plasma samples showed a very homogenous band pattern. Plasma samples showed protein concentrations in the expected range and there were no concerns on sample quality. The lysis of brain tissue pieces delivered sufficient protein for the study. SDS-PAGE of the three brain lysates showed an acceptable band pattern. 
     Plasma Depletion 
     After Top14-depletion of the 12 plasma samples, the total protein masses available were between 189 and 354 µg. SDS-PAGE of Top14 depleted plasma samples showed a homogenous band pattern. Visual assessment confirmed a typical post-depletion pattern with e.g., albumin bands clearly reduced. about95% depletion was achieved. 
     TMTpro™ Analytical Quality Control 
     Analysis of TMTpro™ labelling reaction efficiency of the TMTpro™ 16plex experiment showed that ~98.2% of N-terminal amino groups were labelled, which indicates that labelling was essentially complete. All mass spectrometry runs of the fractionated samples passed internal quality assessments based on the maximum intensity of total ion chromatograms (TICs), number of MS scans and number of peptide spectral matches (PSMs). 
     Metrics: Quantified Peptides and Proteins 
     We report 39,686 peptide sequences with expression values across all 12 patient-derived samples (Table 3). Of these, 9,337 are phosphorylated and associated with combinations of 6,047 distinct, confidently localised phosphosites. The 39,686 peptides are associated with 4,192 distinct protein groups, quantified across all 12 samples. Overall, the application of the TMTcalibrator™ phosphoproteomic workflow to human plasma has provided excellent coverage of the plasma proteome and phosphoproteome.  
     
       
         
          TABLE 5
           
               
               
               
               
               
             
               
                 Numbers of detected and quantified features 
               
               
                   
                 Detected 
                 Quantified 
               
               
                 Total # unique 
                 TMT Plex 
                 All samples 
                 All samples 
               
               
                 ≥1 analytical sample 
                 all analytical samples 
                 all analytical samples 
               
             
            
               
                 Peptides 
                 46,346 
                 46,304 
                 35,309 
                 39,686 
               
               
                 (Phosphopeptides) 
                 (12,525) 
                 (12,504) 
                 (7,118) 
                 (9,337) 
               
               
                 Phosphosites 
                 7,578 
                 7,578 
                 4,763 
                 6,047 
               
               
                 Protein groups 
                 4,553 
                 4,552 
                 3,972 
                 4,192 
               
            
           
         
       
     
     In Table 5, the Peptides row represents both non-phosphorylated and phosphorylated peptides, with the latter described in the subsequent row (italicised). Note that Quantified features allows for a certain number of missing values, which are imputed during data pre-processing. 
     Feature Selection: Phosphopeptides 
     We saw a strong up-regulation of phosphopeptides in the AD group compared to controls, with many phosphopeptides associated with expression changes greater than 6-fold. At the applied thresholds, no significantly down-regulated phosphopeptides were detected. 
     The most highly regulated phosphopeptide was the peptide kVIYSQP[p]sAR (logFC:6.6, Contrast #1) from Junctional adhesion molecule A (F11R, also known as JAM-1), which contains a modification on serine 284. The next most highly regulated phosphopeptide was kVD[p]sLkk (logFC:6.5, Contrast #1) from Caveolae-associated protein 2 (CAVIN2, also known as SDPR), which was modified on Serine 241. The third most highly regulated phosphopeptide was vkE[p]sITR (logFC: 6.4, Contrast #1) - a sequence shared between Filamin A (FLNA) and Filamin-B (FLNB), and modified on Serine 2143 (FLNA) and Serine 2098 (FLNB). 
     Looking at the four FLNA phosphopeptide features quantified ( FIGS.  7 A,  7 B,  7 C, and  7 D ), phosphorylated FLNA appears to be more highly abundant in AD compared to the total control group. However, there are some differences in expression between and the young and elderly controls. The “RAPSVAN” peptide containing pS2152 is seen at similar levels in AD and young controls but much lower in cognitively healthy elderly controls. Conversely, peptide “CSGPG” containing pS1459 is high in AD and elderly controls and much lower in the young controls. 
     We also performed post hoc analyses to determine whether phosphopeptides selected by their significance of expression changes could explain differences between experimental classes ( FIGS.  8 A,  8 B, and  8 C ). For all comparisons, selected phosphopeptides were able to successfully distinguish AD and Control groups, indicating that their inter-group expression differences accounted for about 95% (PC1) of the total variance in the dataset. Based on these results, this set of peptides was not associated with significant differences between the two control groups (PC2 &lt; 2%). These results were reflected in the heatmaps, where control groups were mixed on the sample-clustering dendrograms ( FIG.  4   ), while AD groups formed a clear sub-cluster, with significantly higher expression values. 
     Feature Selection: Proteins 
     All contrasts revealed a strong up-regulation of proteins in the disease group compared to controls, with many proteins associated with expression changes greater than 4-fold. At the applied thresholds, only two significantly down-regulated proteins were detected. 
     Across all three contrasts, we observed a good degree of consistency among proteins, both in terms of their identities and fold change magnitude. The most highly regulated protein was Bridging integrator 2 (BIN2) (logFC:4.5, Contrast #1). The next most highly regulated protein was Integrin alpha-IIb (lTGA2B) (logFC:4.5, Contrast #1). The third most highly regulated protein was Protein S100-A12 (S100A12) (logFC: 3.6, Contrast #1). Of the three highly-regulated phosphopepetides described previously, the corresponding total protein expression (FLNA and CAVIN2) was associated with significantly increased levels in the disease samples (FLNA logFC: 1.8, Contrast #1; CAVIN2 logFC: 1.9, Contrast #1). 
     A closer look at the expression profiles between the three observed sample groups for the protein of interest FLNA ( FIG.  2   ) revealed the same trend at the protein level as previously observed at the phosphopeptide level: FLNA expression was higher in the AD group compared to the controls. The comparison of the two control groups in itself showed similar expression with a slightly higher mean for the Young Cognitive Intact group compared to the Elderly Control group. 
     Summary of FLNA Coverage 
     Filamin A (FLNA), was successfully detected at the protein and the phosphopeptide level and was found to be significantly regulated in the comparison of AD patients with both elderly and young controls. The protein itself was quantified in standard searching for tryptic peptides by 85 PSMs from 46 peptides (Table 6) excluding the phosphopeptides. Of the 5 detected phosphopeptides, 4 could be quantified across all individuals (see, e.g., Table 7). The phosphopeptide aP[p]sVANVGSHcDLSLk did not deliver a sufficient number of data points for the control samples as signals of these were below the detectable range. However, when plotting reporter ion intensity values of the respective peptide spectral match it becomes evident that this peptide is nicely detected in the AD samples as opposed to the controls, consistent with the data generated for the peptide RAP[p]sVANVGSHcDLSLk that is covering the same phosphorylation site in the protein (S2152) ( FIG.  9 B ).  
     
       
         
          TABLE 6
           
               
               
               
               
               
               
               
               
               
             
               
                 Quantified peptides (non-phosphorylated) of FLNA 
               
               
                 Protein Description 
                 Number PSMs 
                 Number Peptides 
                 Contrast #1 
                 Contrast #2 
                 Contrast #3 
               
               
                 logFC 
                 adj. p-value 
                 logFC 
                 adj. p-value 
                 logFC 
                 adj. p-value 
               
             
            
               
                 Filamin A 
                 85 
                 46 
                 1.78 
                 6.95E-07 
                 1.68 
                 9.89E-07 
                 1.73 
                 1.03E-07 
               
            
           
         
       
     
     
       
         
          TABLE 7
           
               
               
               
               
               
               
               
               
             
               
                 Phosphopeptides of Target Protein FLNA 
               
               
                 Amino acid sequence 
                 Phosphosite 
                 Contrast #1 
                 Contrast #2 
                 Contrast #3 
               
               
                 logFC 
                 adj. p-value 
                 logFC 
                 adj. p-value 
                 logFC 
                 adj. p-value 
               
             
            
               
                 vkE[p]sITR 
                 pS2143 
                 6.38 
                 1.80E-05 
                 5.47 
                 2.45E-05 
                 5.92 
                 2.14E-06 
               
               
                 aP[p]sVANVGSHcDLSLk 
                 pS2152 
                 Not quantified, as too many missing values in control samples. 
               
               
                 cSGPGL[p]sPGMVR 
                 pS1459 
                 0.21 
                 5.30E-01 
                 0.96 
                 3.25E-03 
                 0.59 
                 1.77E-02 
               
               
                 iPEISIQDMTAQV[?p]t[?p]PSGK 
                 pT2179 or pS2180 
                 1.52 
                 8.51E-02 
                 1.71 
                 5.28E-02 
                 1.62 
                 2.21E-02 
               
               
                 rAP[p]sVANVGSHcDLSLk 
                 pS2152 
                 0.57 
                 4.47E-01 
                 -0.17 
                 8.54E-01 
                 0.20 
                 7.52E-01 
               
            
           
         
       
     
     Overview of phosphopeptides with determined phosphosites of the target protein FLNA. Site positions that are ambiguously localized, with a confidence score below the set threshold (75%), are indicated by the “?” character. Whereas the strong regulation of FLNA peptides is encouraging, there is a risk that signals arise due to post-collection artifacts e.g. platelet activation since AD and control plasma samples were prepared differently. To explore this, and provide an expanded panel of biomarkers to control for artefactual signals, we performed further experiments as set out in Examples 2 and 3. 
     Example 2: Analysis of Changes in FLNA, Phosphorylated FLNA and Other Proteins in Histopaque® 1077 Plasma Samples 
     In this example we used fresh plasma samples from AD and control cohorts prepared by an identical process expected to trigger platelet activation. Again, 6 samples from controls (3 elderly controls, 3 young cognitive intact) (FLNA band negative or ‘-’) and 6 AD patients (FLNA band positive or ‘+’)) were analysed using TMTcalibrator™ with the same AD brain trigger as described in Example 1. Whole blood from AD and control cohorts was collected and processed using the Histopaque® 1077 process described previously. 
     Sample Processing 
     After thawing of plasma samples, all processing steps were performed as described in Example 1. Mass spectrometry was performed using an inclusion list for all FLNA peptides detected in Example 1. 
     Results 
     Using a dedicated Filamin A inclusion scheme during MS acquisition, we obtained good coverage of the Filamin A sequence (approx. 40%). No significant changes in abundance could be observed for the protein as a whole, although particular parts of the sequence showed varying changes in abundance across the different groups. We also only observed subtle differences in expression levels of two of the quantified phosphosites. 
     In addition to FLNA, we quantified a total of 29,786 peptides and 3,297 phosphosites derived from 3,493 proteins, providing a useful resource for the identification of Alzheimer’s disease pathology dependent plasma markers. The statistical analysis indicated the existence of panels of phosphopeptide and protein features driving the separation of the experimental classes and these represent candidate biomarkers independent of platelet activation status. The functional enrichment indicated changes in the cytoskeletal organization, complement cascade regulation and lipoprotein metabolism. 
     FLNA Coverage 
     FLNA was identified with 220 PSMs for 77 (phospho)peptides and quantified in the standard search for tryptic and semi-tryptic peptides by 158 PSMs from 54 peptides (Table 4) excluding the phosphopeptides. All of the four detected phosphopeptides could be quantified across all individuals (Table 5 contains statistical data). Two peptides were only identified in control samples: AGQSAAGAAPGGGVDTR (aa 8 - 24, SEQ ID NO: xx) was identified in 4 out of 6 controls, 3 young and 1 elderly; and DNGNGTYSCSYVPR (SEQ ID NO: vv) was identified in 1 young control only. One peptide, AVPTGDASK (aa 1636 - 1644; SEQ ID NO:CC), was exclusively identified in Alzheimer’s disease samples, with values obtained for 5 out of 6 patients. 
     Surprisingly, when the level of platelet activation was likely to be similar in AD and control samples due to the use of Histopaque® 1077 preparations, the elevation previously seen in AD was lost. In fact, the AD samples had lower levels of free plasma FLNA suggesting AD platelets were less prone to activation than platelets from cognitively healthy controls. We believe this loss of regulation is due to significant release of FLNA in the control plasma samples in this study.  
     
       
         
          TABLE 8
           
               
               
               
               
               
               
               
               
               
               
               
               
             
               
                 Quantification of FLNA in Histopaque® 1077 plasma samples 
               
               
                 Protein Descriptions 
                 Number PSMs 
                 Number Peptides 
                 AD vs Old 
                 AD vs Young 
                 AD vs All Control 
               
               
                 logFC 
                 p-value 
                 adj. p-value 
                 logFC 
                 p-value 
                 adj. p-value 
                 logFC 
                 p-value 
                 adj. p-value 
               
             
            
               
                 Filamin A 
                 158 
                 54 
                 -0.26 
                 5.08E-01 
                 8.49E-01 
                 -0.09 
                 8.11E-01 
                 9.50E-01 
                 -0.18 
                 5.77E-01 
                 8.69E-01 
               
            
           
         
       
     
     We also identified four phosphorylated FLNA peptides, three of which were seen in Example 1 and one which was unique to the current analysis. Unlike the previous study, levels of the two peptides containing pS2152 did not show a regulation between the AD and control groups, although there was a trend for higher expression of the “RAPSVAN” peptide in AD and elderly controls suggesting this can be more related to disease than sample processing, at least in the periphery. Conversely, the peptide “CSGPG” containing pS1459 showed a strong up-regulation in healthy elderly that can be indicative of a protective post-translational modification. Interestingly, this was also seen in the previous study, although there was also an elevation in the AD group which was not seen this time and can instead suggest this site is produced artefactually, requiring further evaluation. The threonine phosphosite at residue T2336 showed a similar behaviour as the aforementioned serine phosphorylation: the levels detected in the elderly control were higher compared to both the Alzheimer’s disease patients, as well as the young healthy controls.  
     
       
         
          TABLE 9
           
               
               
               
               
               
               
               
               
               
             
               
                 Quantified phosphorylated peptides from FLNA in Histopaque® 1077 plasma 
               
               
                 Protein 
                 ptmRS 
                 Global Position 
                 Safe Site 
                 Squat Sequence 
                 PSMs 
                 AD vs CTRL 
               
               
                 logFC 
                 p-value 
                 adj. p-value 
               
             
            
               
                 Filamin A 
                 S3(Phospho): 100 
                 P21333:S2152 
                 S3 
                 AP[p]SVANVGSHCDLSLK 
                 1 
                 -0.26 
                 6.32E-01 
                 8.86E-01 
               
               
                 Filamin A 
                 S7(Phospho): 100 
                 P21333:S1459 
                 S7 
                 CSGPGL[p]SPGMVR 
                 2 
                 -0.41 
                 8.55E-02 
                 5.07E-01 
               
               
                 Filamin A 
                 S4(Phospho): 100 
                 P21333:S2152 
                 S4 
                 RAP[p]SVANVGSHCDLSLK 
                 1 
                 -0.10 
                 8.58E-01 
                 9.64E-01 
               
               
                 Filamin A 
                 T3(Phospho): 100 
                 P21333:T2336 
                 T3 
                 RL[p]TVSSLQESGLK 
                 1 
                 -0.63 
                 2.22E-01 
                 6.64E-01 
               
            
           
         
       
     
     Example 3: TMTcalibrator™ Analysis of AD and Control EDTA Plasma Samples 
     The results of Example 2 provided evidence that some regulation of protein expression can be driven by the sample preparation method. We therefore completed our study by analysing fresh samples of AD and control plasma samples that had all been prepared from EDTA blood collection tubes under identical conditions. All other conditions were as described in Examples 1 and 2. We expected the level of platelet activation to be the lowest in this set of samples, which better reflect standard clinical practice. 
     Results 
     FLNA was successfully detected at the protein and phosphopeptide level. We detected 60 unmodified peptides associated with FLNA, of which 54 were unique to FLNA and 42 could be quantified in all 12 samples. The protein was found to be significantly upregulated (about2-fold) in the AD samples compared to the healthy control groups. Three of four detected phosphopeptides associated with FLNA were used for quantification. These peptides carry modifications at the phosphorylation sites pS1459, pS2143 and pS2152. All three phosphopeptide features were more abundant in the AD groups, as was seen in Example 1, but the extent of regulation was not as strong here. We hypothesize that this reflects the true biological difference in FLNA abundance in patients with Alzheimer’s disease. Where platelet activation is significant, the amount of released FLNA is sufficient to mimic that seen in disease and even reverse the relative abundance signature between healthy individuals and AD patients.  
     
       
         
          TABLE 10
           
               
               
               
               
               
               
               
               
               
             
               
                 Relative expression of phosphorylated FLNA peptides in EDTA plasma 
               
               
                 Protein 
                 ptmRS 
                 Global Position 
                 Safe Site 
                 Squat Sequence 
                 PSMs 
                 AD vs CTL 
               
               
                 logFC 
                 p-value 
                 adj. p-value 
               
             
            
               
                 Filamin A 
                 S4(Phospho): 100 
                 P21333:S2143 |O7 5369.S2098 
                 S4 
                 vkE[p]sITR 
                 2 
                 2.76 
                 4.32E-04 
                 2.79E-02 
               
               
                 Filamin A 
                 S3(Phospho): 100 
                 P21333:S2152 
                 S3 
                 aP[p]sVANVGSHcDLSLk 
                 2 
                 0.87 
                 4.84E-03 
                 1.02E-01 
               
               
                 Filamin A 
                 S7(Phospho): 100 
                 P21333:S1459 
                 S7 
                 cSGPGL[p]sPGMVR 
                 3 
                 1.42 
                 1.22E-02 
                 1.57E-01 
               
            
           
         
       
     
     
       
         
          TABLE 11
           
               
               
               
               
               
               
               
               
               
               
               
               
             
               
                 Relative expression of non-phosphorylated FLNA in EDTA plasma 
               
               
                 Protein 
                 #PSMs 
                 #Peptides 
                 AD vs Old 
                 AD vs Young 
                 AD vs All Control 
               
               
                 logFC 
                 p-value 
                 adj. p-value 
                 logFC 
                 p-value 
                 adj. p-value 
                 logFC 
                 p-value 
                 adj. p-value 
               
             
            
               
                 Filamin A 
                 116 
                 42 
                 0.96 
                 2.19E-08 
                 3.81E-05 
                 1.01 
                 1.29E-08 
                 4.4810-05 
                 0.98 
                 4.55E-09 
                 7.92E-06 
               
            
           
         
       
     
     In addition to FLNA, we quantified a total of 31,382 peptides, 7,677 phosphopeptides and 3,478 proteins. The statistical analysis revealed multiple significantly regulated phosphopeptide and protein features driving the separation of the experimental classes. The functional enrichment indicated changes in the platelet activation, cell-extracellular matrix interaction and cell junction organization. 
     We obtained good characterization of Filamin A in AD and control K2EDTA plasma. Unlike the results obtained with plasma prepared with the Histopaque® 1077 method, quantitative differences in Filamin A abundance and multiple other protein features were detected in the current study. The results were comparable to the first study (40-224), albeit the observed differences were less pronounced. 
     We found various FLNA-associated tryptic peptides with good regulation between the AD and control groups. We believe that these results can serve as good basis to develop a targeted mass spectrometry method for FLNA with diagnostic and prognostic utility in the context of selection and monitoring of patients for treatment with simufilam. 
     Example 4: Comparison of Protein Expressions in AD and Cognitively Normal Individuals Across Three Different Sample Preparation Methods 
     We compared the relative expression of different proteins, including FLNA and phosphorylated peptides of FLNA in the different plasma groups of AD Histopaque® 1077 vs Control EDTA, AD Histopaque® 1077 vs Control Histopaque® 1077, and AD EDTA vs Control EDTA. This confirmed the suitability of FLNA, and in particular phosphorylated eptiopes of FLNA to serve as peripheral biomarkers of Alzheimer’s disease. However, it is evident that the potential activation of platelets using non-standard plasma preparation can potentially mimic the elevated FLNA levels seen in AD and we therefore searched for peptides and proteins whose levels were elevated in Histopaque® 1077 plasma but were not detected in EDTA plasma. We also focused on a specific subset of such proteins with documented associations with platelet biology. 
     The relative expression of FLNA is clearly affected by the use of Histopaque® 1077 for plasma preparation. In general, we see that the relative level of phosphorylated FLNA is particularly increased in cognitively healthy individuals compared to AD patients (Table 12). In Example 1 Histopaque® 1077 was used to prepare AD plasma whereas controls were prepared using EDTA. All three of the phosphorylated FLNA peptides detected in Example 1 were found increased in the AD group, but two of these showed a reduced level in AD vs controls when both were prepared by Histopaque® 1077. For pS1459 of FLNA the extent of regulation in AD vs control groups was seen to be further extended in EDTA-prepared plasma samples tested in Example 3.  
     
       
         
          TABLE 12
           
               
               
               
               
               
             
               
                 Relative expression of FLNA phosphopeptides in plasma from AD patients compared to controls from Examples 1 - 3 
               
               
                   
                 Log2 Fold-change AD vs CTL 
               
               
                 Peptide Sequence 
                 Phosphorylated Residue 
                 Example 1 
                 Example 2 
                 Example 3 
               
             
            
               
                 cSGPGL[p]sPGMVR 
                 S1459 
                 0.586 
                 -0.407 
                 1.421 
               
               
                 vkE[p]sITR 
                 S2143 
                 N D 
                 ND 
                 2.758 
               
               
                 aP [p] sVANVGSHcDLSLk 
                 S2152 
                 ND 
                 -0.263 
                 0.874 
               
               
                 rAP[p]sVANVGSHcDLSLk 
                 S2152 
                 0.2.02 
                 -0.100 
                 ND 
               
               
                 iPEISIQDMTAQV[?p] t [?p] sPSGk 
                 T2179?; S2180? 
                 1.619 
                 ND 
                 ND 
               
               
                 rL [p] tVSSLQESGLk 
                 T2336 
                 ND 
                 -0.625 
                 ND 
               
            
           
         
       
     
     We also identified three peptides that could be used to assess the level of artefactual platelet activation during preparation of plasma or serum. These peptides were derived from proteins with annotations to roles in platelet biology, and were differentially expressed in AD vs control groups when Histopaque® 1077 was used in the AD samples only, were not regulated when Histopaque® 1077 was used for both cohorts, and were not detected in either group when EDTA was used to prepare plasma. In this respect, the inclusion of these peptides or their respective proteins in methods of the present invention can be used to indicate samples where platelet activation has occurred and a fresh sample should be tested where the absence of these peptides/proteins is indicative of a good sample.  
     
       
         
          TABLE 13
           
               
               
               
               
               
             
               
                 Peptides indicating excessive platelet activation 
               
               
                   
                   
                 Log2 Fold-change AD vs Control 
               
               
                 Peptide Sequence 
                 Protein 
                 Example 1 
                 Example 2 
                 Example 3 
               
             
            
               
                 aEYSPcR 
                 Integrin alpha-IIb 
                 4.41 
                 -0.28 
                 ND 
               
               
                 gALHDENTcNR 
                 Integrin beta-3 
                 5.33 
                 0.00 
                 ND 
               
               
                 eYVNV [p] sQEL HPGAAk 
                 Linker for activation of T-cells family member 1 
                 3.35 
                 -1.08 
                 ND 
               
            
           
         
       
     
     Throughout this specification, various patents, patent applications and/or other types of publications (e.g., journal articles and books) are referenced. The disclosure of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety for all purposes.