BLOOD-BASED DIAGNOSTIC ASSAYS FOR ALZHEIMER'S DISEASE
20230144446 · 2023-05-11
Inventors
Cpc classification
G01N2800/56
PHYSICS
A61P25/28
HUMAN NECESSITIES
G01N2800/52
PHYSICS
International classification
Abstract
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.
Claims
1. A biomarker panel comprising one or more phosphorylated peptides obtained from in vitro digestion of a Filamin A protein having the polypeptide sequence of SEQ ID NO: 1.
2. The biomarker panel of claim 1, wherein the Filamin A is contained within or obtained from a biological fluid or tissue sample taken from a subject suspected of having a neurological disease.
3. The biomarker panel of claim 3, wherein the neurological disease is Alzheimer’s disease.
4. The biomarker panel of claim 1, wherein the phosphorylated peptide is phosphorylated at a residue corresponding to serine 2152 of SEQ ID NO: 1.
5. The biomarker panel of claim 1, wherein 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.
6. The biomarker panel of claim 1, wherein the biomarker panel comprises a plurality of phosphorylated fragments of FLNA, optionally two or more fragments or three or more fragments.
7. The biomarker panel of claim 1, wherein the biomarker panel comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different phosphorylated fragments of FLNA.
8. The biomarker panel of claim 1, wherein the biomarker panel comprises 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.
9. The biomarker panel of claim 1, wherein the phosphorylated peptide is phosphorylated at a position corresponding to serine 2143 of SEQ ID NO: 1.
10. The biomarker panel of claim 1, wherein 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.
11. The biomarker panel of claim 1, wherein the biomarker panel comprises two or more phosphorylated peptides, each comprising a fragment of FLNA and having the amino acid sequence of any one 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.
12. The biomarker panel of claim 1, wherein the phosphorylated peptide is phosphorylated at a position corresponding to serine 2180 of SEQ ID NO: 1.
13. The biomarker panel of claim 1, wherein the phosphorylated peptide has the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.
14. The biomarker panel of claim 1, wherein the biomarker panel comprises two or more phosphorylated peptides, each comprising a fragment of FLNA and having the amino acid sequence of any one of SEQ ID NOs: 10-12, wherein at least one of the two or more phosphorylated peptides is phosphorylated at a position corresponding to serine 2180 of SEQ ID NO: 1.
15. A biomarker panel, wherein the biomarker panel comprises a plurality of phosphorylated peptides, wherein each phosphorylated peptide is a fragment of SEQ ID NO:1 and comprises a phosphorylation site at a position corresponding to serine 2143, 2152, and/or 2180 of SEQ ID NO: 1.
16. A biomarker panel, comprising a plurality of phosphorylated peptides, each having the sequence of SEQ ID Nos: 2-12 and being phosphorylated at a position corresponding to serine 2143, 2152, and/or 2180 of SEQ ID NO: 1.
17. A biomarker panel, comprising 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 2143, 2152 and/or 2180 of SEQ ID NO: 1.
18. A biomarker panel, 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 1-4.
19. A method for the diagnosis/prognosis of a neurological disorder, comprising: obtaining a bodily fluid or tissue sample from a subject; digesting one or more proteins in the bodily fluid or tissue sample with one or more proteases; and detecting and/or measuring the level of one or more peptides produced from the digestion, using mass spectrometry, wherein the one or more peptides are obtained from in vitro digestion of one or more of the proteins listed in Tables 1-4.
20. A method for monitoring the progression of a neurological disorder, comprising: (a) obtaining a bodily fluid or tissue sample from a subject at a first time point; (b) digesting one or more proteins in the bodily fluid or tissue sample with one or more proteases; (c) detecting and/or measuring the level of one or more peptides produced from the digestion, using mass spectrometry, wherein the one or more peptides are obtained from in vitro digestion of one or more of the proteins listed in Tables 1-4; and (d) repeating steps (a)-(c) at a second time point.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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).
[0074] 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.
[0075] 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).
[0076] 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) >350 ng/L, phospho-tau (P-tau) >80 ng/L and A~42 <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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] The term “phosphopeptide” means a peptide that contains at least one amino acid modified by addition of a phosphate group.
[0082] 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.
[0083] As used herein, the term “subject” includes any human or non-human animal.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] The term “comprising” indicates that the subject includes all the elements listed, but can, optionally, also include additional, unnamed elements.
[0090] 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,
[0091] 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.
[0092] 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).
[0093] 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.
[0094] 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.
[0095] 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).
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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-US-00001 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-US-00002 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-US-00003 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-US-00004 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-US-00005 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
[0103] 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
[0104] 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
[0105] For this experiment, a TMTcalibrator™ phosphoproteomic analysis (
[0106] 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
[0107] 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
[0108] Available protein from pooled AD brain tissue lysates was used as a calibrator/trigger brain sample.
Protein Concentration Measurement and SDS-PAGE
[0109] 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
[0110] 35 .Math.L 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 .Math.l each were depleted to obtain sufficient amounts (Table S2).
Digestion and TMTpro Labelling
[0111] 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.
[0112] Analytical depleted plasma samples: 180 .Math.g 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.sup.® tC18 cartridges) samples were lyophilised to dryness.
[0113] 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 .Math.g = 2160 .Math.g) + pooled brain digest mixture (about 3780 .Math.g in 4 channels with a ratio of 1:4:6:10) in a protein mass ratio of 1:1.75). 50 .Math.g 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 .Math.g were taken off as samples for basic-reversed fractionation for the non-enriched arm (including a backup sample) and about 5,690 .Math.g portions for phosphopeptide enrichment.
Phosphopeptide Enrichment
[0114] About 5,690 .Math.g 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)
[0115] bRP fractionation was conducted for the TMTpro™ 16plex taking A) 100 .Math.g 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)
[0116] 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 .Math.m 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 .Math.m 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.
[0117] 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
[0118] In total, 12 separate raw mass spectrometry data files (12 fractions - 6 enriched & 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.
[0119] 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
[0120] 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
[0121] 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.
[0122] 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
[0123] Protein determination by modified Bradford assay showed that protein concentrations of plasma samples were between 41.9 and 73.6 .Math.g/.Math.l (mean: about 59 .Math.g/.Math.l). AD samples showed a lower mean protein concentration versus controls (48.7 vs 68.5 .Math.g/.Math.l), 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
[0124] After Top14-depletion of the 12 plasma samples, the total protein masses available were between 189 and 354 .Math.g. 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
[0125] 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
[0126] 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-US-00006 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
[0127] 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
[0128] 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.
[0129] 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).
[0130] Looking at the four FLNA phosphopeptide features quantified (
[0131] We also performed post hoc analyses to determine whether phosphopeptides selected by their significance of expression changes could explain differences between experimental classes (
Feature Selection: Proteins
[0132] 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.
[0133] 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).
[0134] A closer look at the expression profiles between the three observed sample groups for the protein of interest FLNA (
Summary of FLNA Coverage
[0135] 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) (
TABLE-US-00007 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-US-00008 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
[0136] 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
[0137] 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
[0138] 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
[0139] 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.
[0140] 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
[0141] 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.
[0142] 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-US-00009 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
[0143] 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-US-00010 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
[0144] 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
[0145] 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-US-00011 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-US-00012 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
[0146] 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.
[0147] 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.
[0148] 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
[0149] 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.
[0150] 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-US-00013 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
[0151] 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-US-00014 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
[0152] 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.