ROLE OF CITRULLINATION IN DIAGNOSING DISEASES

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0001] This invention was made with Government support under Grant Nos. HL112586-01 and HHSN268201000032C awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

[0002] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Citrullination, the irreversible post-translational modification (PTM) involving the conversion of arginine to citrulline by the family of enzymes peptidylarginine deiminase (PAD), is associated with several diseases. Citrullination appears to be a generalized process; autoantibodies targeting citrullinated proteins are relatively specific for rheumatoid arthritis (RA) and, although occasionally observed in other autoimmune conditions, are uncommonly observed in healthy individuals. In our previous study, elevated levels of autoantibodies to citrullinated protein antigens were found in the myocardium of RA patients. PADs were detected in cardiomyocytes, resident inflammatory cells, endothelial cells and vascular smooth muscle cells.

[0004] Citrullination results in a small increase in molecular mass (+0.984 Da) but converts the positively charged guanidine group on an arginine residue into the neutrally charged ureido group on the citrulline amino acid. The loss of charge from an arginine to a citrulline can have dramatic consequences on protein structure, proteolytic susceptibility, protein- protein interactions and intracellular signaling. Since citrullination can lead to profound changes in protein structure and function, it is not surprising that citrullination and the PAD enzymes are found in numerous chronic diseases. Furthermore, the conversion of arginine to citrulline is catalyzed in a Ca2+-dependent manner with relatively high intracellular concentration of calcium. Because the cytosolic and nucleoplasmic calcium concentrations are relatively low, PADs should be inactive under normal conditions. However PADs become activated in injured and dying cells, when calcium concentrations increase because of the influx of calcium ions from the extracellular environment and release from intracellular calcium stores.

[0005] The actual proteins that are citrullinated in myocardium is unknown as is whether i) myocardial citrullinated proteins are immune targets for circulating autoantibodies, ii) myocardial citrullinated proteins can themselves induce an autoimmune response, and iii) citrullinated proteins directly mediate phenotypic modifications to cardiac structure or function. There is precedent for PTMs of myocardial proteins leading to changes in cardiac contractility and structure in heart failure (HF). Phosphorylation, oxidation, and acetylation of sarcomeric proteins cause morphologic changes to proteins that lead to decreased contractile performance and adverse cardiac remodeling with HF. However, it is unknown whether citrullination of myocardial proteins plays a similar role.

[0006] The framework of health information on neurodegenerative diseases sometimes includes brain diseases, defined as pathologic conditions also affecting the brain (composed of the intracranial components of the central nervous system). This includes (but is not limited to) the cerebral cortex, intracranial white matter, basal ganglia, thalamus, hypothalamus, brain stem, and cerebellum (MeSH definition). It is important to note that not all individuals with a family history of neurodegenerative disease will have an identifiable gene mutation. This could be because the responsible gene has not yet been identified, or because the disease is not actually due to a gene mutation.

[0007] There have been challenges in identification of citrullinated proteins and the modified amino acid residues. Here, the inventors provide examples of cirtullinated proteins and peptides showing modified amino acid residues which may serve as markers for cardiovascular diseases and neurodegenerative diseases.

SUMMARY

[0008] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions and methods which are meant to be exemplary and illustrative, not limiting in scope

[0009] Provided herein are method for diagnosing and predicting cardiovascular diseases. The methods include obtaining a biological sample from a subject in need of diagnosing cardiovascular disease and detecting the amount of a citrullinated protein or a citrullinated peptide in the biological sample obtained from said subject. In some embodiments a change in the level of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increase risk of cardiovascular disease. In one embodiment, the change in citrullination levels is an increase in the levels of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increased risk of cardiovascular disease. In another embodiment, the change in citrullination levels is a decrease in the levels of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increased risk of cardiovascular disease. In exemplary embodiments, presence of one or more citrullinated proteins or peptides shown in Table 1A, Table IB, Table 1C, Table ID and/or Table IE is indicative of increased risk of cardiovascular disease. In exemplary embodiments, a change in the levels of citrullinated proteins or peptides shown in Table 1 A, Table IB, Table 1C, Table ID and/or Table IE is indicative of increased risk of cardiovascular diseases. In exemplary embodiments, the change in the levels is an increase in the levels of citrullination of proteins or peptides shown in Table 1A, Table IB, Table 1C, Table ID and/or Table IE which is indicative of increased risk of cardiovascular diseases. In exemplary embodiments, the change in the levels is a decrease in the levels of citrullination of proteins or peptides shown in Table 1A, Table IB, Table 1C, Table ID and/or Table IE which is indicative of increased risk of cardiovascular diseases. In some embodiments, an increase in citrullination levels of some peptides shown in Table 1A, Table IB, Table 1C, Table ID and/or Table IE and a decrease in citrullination of some peptides shown in Table 1A, Table IB, Table 1C, Table ID and/or Table IE is indicative of increased risk of cardiovascular disease. In various embodiments, citrullination is at the Arginine residues, as indicated as "Dea" in Table 1A, Table IB, Table 1C, Table ID and/or Table IE. In exemplary embodiments, cardiovascular diseases are any one or more of ischemic heart disease (ISHD), idiopathic myopathy (IDCM) or combinations thereof.

[0010] Also provided herein are methods for treating cardiovascular diseases in a subject in need thereof. The methods include diagnosing cardiovascular diseases by the methods set forth herein and administering an effective amount of a therapeutic agent to the subject diagnosed with cardiovascular diseases so as to treat cardiovascular diseases in the subject. In exemplary embodiments, cardiovascular diseases are any one or more of ischemic heart disease (ISHD), idiopathic myopathy (IDCM) or combinations thereof. [0011] Provided herein are methods for diagnosing and/or predicting amyotropic lateral sclerosis (ALS). The methods include obtaining a biological sample from a subject in need of diagnosing ALS and detecting citrullinated protein or citrullinated peptide in the biological sample obtained from said subject. In some embodiments, presence of citrullinated proteins or citrullnated peptides is indicative of increased risk of ALS. In some embodiments a change in the level of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increased risk of ALS. In one embodiment, an increase in the levels of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increased risk of ALS. In another embodiment, a decrease in the levels of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increased risk of ALS. In an exemplary embodiment, presence of citrullinated peptides shown in Table 2 is indicative of ALS. In exemplary embodiments, presence of one or more citrullinated peptides shown in Tables 2, 3 or 4 is indicative of increased risk of ALS. In exemplary embodiments, a change in the levels of citrullinated peptides shown in Tables 2, 3 or 4 is indicative of increased risk of ALS. In exemplary embodiments, a change in the amount of citrullination of peptides shown in Tables 2, 3 or 4 is indicative of increased risk of neurodegenerative diseases. In various embodiments, citrullination is at the Arginine residues, as indicated as "Dea" in Table 2, 3 or 4.

[0012] Also provided herein are methods for treating ALS in a subject in need thereof. The methods include diagnosing ALS by the methods set forth herein and administering an effective amount of a therapeutic agent to the subject diagnosed with ALS so as to treat ALS in the subj ect.

[0013] Provided herein are methods for diagnosing and/or predicting chronic neurodegenerative disease. The methods include obtaining a biological sample from a subject in need of diagnosing chronic neurodegenerative disease and detecting citrullinated proteins or citrullinated peptides in the biological sample obtained from said subject. In some embodiments, presence of citrullinated proteins or citrullinated peptides is indicative of increased risk of chronic neurodegenerative disease. In some embodiments a change in the level of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increased risk of chronic neurodegenerative disease. In one embodiment, an increase in the levels of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increased risk of chronic neurodegenerative disease. In another embodiment, a decrease in the levels of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increased risk of chronic neurodegenerative disease. In exemplary embodiments, a change in the levels of one or more citrullinated proteins or citrullinated peptides shown in Tables 5A-5B is indicative of increased risk of neurodegenerative diseases. In exemplary embodiments, the change in the cirullination levels is an increase in the levels of citrullination of one or more peptides or proteins shown in Tables 5A-5B which is indicative of increased risk of neurodegenerative diseases. In exemplary embodiments, the change in the citrullination levels is a decrease in the levels of citrullination of one or more peptides or proteins shown in Tables 5A-5B which is indicative of increased risk of chronic neurodegenerative diseases. In some embodiments, an increase in citrullination levels of some peptides shown in Table 5A-5B and a decrease in citrullination of some peptides shown in Tables 5A-5B is indicative of increased risk of chronic neurodegenerative disease. In exemplary embodiments, examples of neurodegenerative diseases include but are not limited to acquired brain injury, chronic neurodegenerative disease Alzheimer disease, Parkinson disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington disease, Friedreich's ataxia, spinal muscular atrophy, prion diseases, multiple sclerosis, stroke, degenerative nerve diseases, encephalitis, spinal muscular dystrophy and/or Creutzfeld- Jakob disease.

[0014] Also provided herein are methods for treating chronic neurodegenerative diseases in a subject in need thereof. The methods include diagnosing chronic neurodegenerative diseases by the methods set forth herein and administering an effective amount of a therapeutic agent to the subject diagnosed with chronic neurodegenerative disease so as to treat chronic neurodegenerative disease in the subject. In exemplary embodiments, examples of neurodegenerative diseases include but are not limited to acquired brain injury, chronic neurodegenerative disease Alzheimer disease, Parkinson disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington disease, Friedreich's ataxia, spinal muscular atrophy, prion diseases, multiple sclerosis, stroke, degenerative nerve diseases, encephalitis, spinal muscular dystrophy and/or Creutzfeld- Jakob disease.

[0015] Provided herein are methods for diagnosing and/or predicting acquired brain injury. The methods include obtaining a biological sample from a subject in need of diagnosing acquired brain injury and detecting citrullinated proteins or citrullinated peptides in the biological sample obtained from said subject. In some embodiments, presence of citrullinated proteins or citrullinated peptides is indicative of increased risk of acquired brain injury. In some embodiments a change in the level of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increased risk of acquired brain injury. In one embodiment, an increase in the levels of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increased risk of acquired brain injury. In another embodiment, a decrease in the levels of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increased risk of acquired brain injury. In exemplary embodiments, presence of one or more citrullinated proteins or citrullinated peptides shown in Tables 6A-6B is indicative of increased risk of acquired brain injury. In exemplary embodiments, a change in the levels of one or more citrullinated proteins or citrullinated peptides shown in Tables 6A-6B is indicative of increased risk of acquired brain injury. In exemplary embodiments, the change in the levels of citrullination is an increase in the levels of one or more peptides or proteins shown in Tables 6A-6B which is indicative of increased risk of acquired brain injury. In exemplary embodiments, the change in the citrullination levels is a decrease in the levels of citrullination of one or more peptides or proteins shown in Tables 6A-6B which is indicative of increased risk of acquired brain injury. In some embodiments, an increase in citrullination levels of some peptides shown in Table 6A-6B and a decrease in citrullination of some peptides shown in Tables 6A-6B is indicative of increased risk of acquired brain injury. In exemplary embodiments, acquired brain injury includes but is not limited to traumatic brain injuries (TBI's), strokes, brain illness, tumor, hemorrhage, and any other kind of brain injury acquired after birth.

[0016] Also provided herein are methods for treating acquired brain injury in a subject in need thereof. The methods include diagnosing acquired brain injury by the methods set forth herein and administering an effective amount of a therapeutic agent to the subject diagnosed with acquired brain injury so as to treat acquired brain injury in the subject. In exemplary embodiments, acquired brain injury includes but is not limited to traumatic brain injuries (TBI's), strokes, brain illness, tumor, hemorrhage, and any other kind of brain injury acquired after birth.

BRIEF DESCRIPTION OF THE FIGURES

[0017] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive

[0018] Figure 1 depicts in accordance with various embodiments of the invention, the cardiac citrullinated proteome. Diagrams show citrullinated proteins with significant p value group by (A) cellular component and by (B) molecular function. Details can be found in Tables 1A-F. (C) Detection of citrullinated proteins in heart obtained from control and HF patients (ISHD, IDCM) using 4-12% SDS PAGE. Densitometry analysis revealed that there was no significant change between groups.

[0019] Figure 2 depicts in accordance with various embodiments of the invention, 2DE DIGE analysis with samples treated with PAD2. (A) Samples labeled with Cy2 (internal control), Cy3 (untreated) and Cy5 (treated) as described in methods section. (B) Proteins described by the network: contractility and energy production. Citrullinated proteins up and down regulated in ischemia were mapped onto the human STRING database of known human protein interactions. Red, protein marked as down regulated, green, proteins up- regulated and blue = protein-protein interacting partner. The following abbreviations were used: FLNC: Filamin C; LDB3 : LIM domain-binding protein 3; MYBPC: Myosin-binding protein C, cardiac-type; MYOZ2: Myozenin-2; TNNI3 : Troponin I; MYH: Myosin; CRYAB: Alpha-crystallin B chain; VFM: ATP synthase subunit alpha; AHNAK: Neuroblast differentiation-associated protein; HSP90AA1 : Heat shock protein HSP 90-alpha; HSPD1 : 60 kDa heat shock protein; PPIA: Peptidyl-prolyl cis-trans isomerase A; FABP3 : Fatty acid- binding protein.

[0020] Figure 3 depicts in accordance with various embodiments of the invention, actin- binding studies. Increasing concentrations of A) TM (0.5uM to 2 uM) were incubated with F-actin or B) actin-HMM and C) Tnl (0.5uM to 2uM) in buffer containing 40mM Tris-HCl (pH 7.6), lOOmM NaCl, 5 mM MgC12, and 1 mM DTT. Binding of TM to F-actin was carried out at 25°C for 30 min and ultracentrifuged at 60,000 rpm for 25 min, 20°C, in a Beckman model TL- 100.2. Both pellet and supernatant (unbound protein) were analyzed. Representative silver-stained gels show proteins composition of the supernatants and pellets. A triplicate set of gels, were analyzed by densitometry. Each data point is an average (and range) of the values obtained from the three sets of gels.

[0021] Figure 4 depicts in accordance with various embodiments of the invention, citrullination of sarcomeric proteins, biochemical and physiological effects. A) Regulation of the actomyosin HMM-ATPase activity by citrullinated F-actin and/or citrullinated HMM. B) Inhibition of actomyosin HMM-ATPase activity by TM. ATPase activity was measured as a function of TM concentration. The results are the average of four independent experiments for each protein at each TM concentration. Assay conditions: 0.2mg/ml F-actin, 0.02mg/ml HMM, 0-2.0 uM TM in 10 mM Hepes, pH 7.5, 30 mM NaCl, 5 mM MgC12, 4 mM ATP. PAD2 treatment reduced myofilament calcium sensitivity. C) Force-calcium relationships for untreated membrane-permeabilized myocytes from untreated control (n=8 myocytes from 3 mice, gray circles) and PAD2 treated (n=8 myocytes from 3 mice, open circles) groups. D) There was no difference in maximal calcium activated force (Fmax) between the two groups. E) PAD2 treatment caused a significant (p=0.009) increase in EC50 (calcium required to generate 50% Fmax), indicating a decrease in calcium sensitivity. F) While the hill coefficient (nH) trended to be decreased by PAD2 treatment (see steepness of curve in panel C), the difference was not significant (p=0.34).

[0022] Figure 5 depicts in accordance with various embodiments of the invention, RT-PCR analysis of expression level of PAD isoforms in A, B) heart from control mouse, C) mouse keratinocytes and D) mouse macrophage cell line activated by lipopolysaccharide. The PCR product PAD2 is seen in all types of samples; PAD4 and PAD1 is seen in cardiac fibroblast, keratinocytes and macrophage cell line. PAD3 has not been detected. (MW: PAD1 285bp; PAD2 390bp; PAD3 200bp; PAD4 550bp).

[0023] Figure 6 depicts in accordance with various embodiments of the invention, a citrullinated protein up/down regulated between heart failure groups, ischemia and IDCM.

[0024] Figure 7 depicts in accordance with various embodiments of the invention, citrullination of the contractile proteins could affect different aspects of regulatory function. It could either trigger a structural change or stabilizes a conformation that is necessary for actin-activated release of Pi and completion of the ATPase cycle.

[0025] Figure 8 depicts in accordance with various embodiments of the invention, the use of SWATH-MS technology to identify citrullination of protein involved in neurodegenerative diseases.

[0026] Figure 9 depicts in accordance with various embodiments of the invention, an overview bioinformatics workflow for processing citrullinated spectra library file.

DETAILED DESCRIPTION

[0027] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (September 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3rd ed., revised ed., J. Wiley & Sons (New York, NY 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2nd ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U. S. Patent No. 5,585,089 (1996 Dec); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7.

[0028] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention. Indeed, the present invention is in no way limited to the methods and materials described. For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0029] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [0030] The terms "marker" or "biomarker" are used interchangeably herein, and in the context of the present invention refer to a protein or peptide that has specific citrullinated amino acid residues or the enzyme itself, PAD 1, PAD2, PAD3 or PAD4 (of a particular specific identity or apparent molecular weight) which is differentially present in a sample taken from patients having a specific disease or disorder as compared to a control value, the control value consisting of, for example, average or mean values in comparable samples taken from control subjects (e.g., a person with a negative diagnosis, normal or healthy subject). Biomarkers may be determined as specific peptides or proteins (Tables 1-10) which may be detected by antibodies or mass spectroscopy. In some applications, for example, a mass spectroscopy or other profile or multiple antibodies may be used to determine multiple biomarkers, and differences between individual biomarkers and/or the partial or complete profile may be used for diagnosis. This can include detection of the enzyme or a protein it has citrullinated, alone or in combination.

[0031] The term "differentially present" or "change in level" refers to differences in the quantity and/or the frequency of a marker present in a sample taken from patients having a specific disease or disorder as compared to a control subject. For example, a marker can be present at an elevated level or at a decreased level in samples of patients with the disease or disorder compared to a control value (e.g. determined from samples of control subjects). Alternatively, a marker can be detected at a higher frequency or at a lower frequency in samples of patients compared to samples of control subjects. A marker can be differentially present in terms of quantity, frequency or both as well as a ratio of differences between two or more specific modified amino acid residues and/or the enzyme itself. In one embodiment, an increase in the ratio of modified to unmodified proteins and peptides described herein is diagnostic of any one or more of the diseases described herein. In an embodiment, an increase in citrullination of proteins and peptides as described herein refers to an increase in ratio of modified (citrullinated) to unmodified (non-citrullinated) proteins and/or peptides described herein. In one embodiment, a decrease in the ratio of modified to unmodified proteins and peptides described herein is diagnostic of any one or more of the diseases described herein. In an embodiment, a decrease in citrullination of proteins and peptides as described herein refers to a decrease in ratio of modified (citrullinated) to unmodified (non- citrullinated) proteins and/or peptides described herein.

[0032] A marker, compound, composition or substance is differentially present in a sample if the amount of the marker, compound, composition or substance in the sample is statistically significantly different from the amount of the marker, compound, composition or substance in another sample, or from a control value. For example, a compound is differentially present if it is present at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000%) greater or less than it is present in the other sample (e.g. control), or if it is detectable in one sample and not detectable in the other.

[0033] Alternatively or additionally, a marker, compound, composition or substance is differentially present between samples if the frequency of detecting the marker, etc. in samples of patients suffering from a particular disease or disorder, is statistically significantly higher or lower than in the control samples or control values obtained from healthy individuals. For example, a biomarker is differentially present between the two sets of samples if it is detected at least about 10%>, at least about 20%, at least about 30%>, at least about 40%), at least about 50%, at least about 60%>, at least about 70%, at least about 80%, at least about 90%, or at least about 100% more frequently or less frequently observed in one set of samples than the other set of samples. These exemplary values notwithstanding, it is expected that a skilled practitioner can determine cut-off points, etc. that represent a statistically significant difference to determine whether the marker is differentially present.

[0034] "Diagnostic" means identifying the presence or nature of a pathologic condition and includes identifying patients who are at risk of developing a specific disease or disorder. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay, are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

[0035] The terms "detection", "detecting" and the like, may be used in the context of detecting biomarkers, or of detecting a disease or disorder (e.g. when positive assay results are obtained). In the latter context, "detecting" and "diagnosing" are considered synonymous.

[0036] By "indicative of or "at risk of is intended to mean at increased risk of, compared to a normal subject, or compared to a control group, e.g. a patient population. Thus a subject carrying a particular marker may have an increased risk for a specific disease or disorder, and be identified as needing further testing. "Increased risk" or "elevated risk" mean any statistically significant increase in the probability, e.g., that the subject has the disorder. The risk is preferably increased by at least 10%, more preferably at least 20%, and even more preferably at least 50% over the control group with which the comparison is being made.

[0037] A "test amount" of a marker refers to an amount of a marker present in a sample being tested. A test amount can be either in absolute amount (e.g., .mu.g/ml) or a relative amount (e.g., relative intensity of signals).

[0038] A "diagnostic amount" of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of a particular disease or disorder. A diagnostic amount can be either in absolute amount (e.g., μg/ml) or a relative amount (e.g., relative intensity of signals).

[0039] A "control amount" of a marker can be any amount or a range of amount which is to be compared against a test amount of a marker. For example, a control amount of a marker can be the amount of a marker in a person who does not suffer from the disease or disorder sought to be diagnosed. A control amount can be either in absolute amount (e.g., μg/ml) or a relative amount (e.g., relative intensity of signals).

[0040] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of .alpha.-amino acid residues, in particular, of naturally-occurring .alpha.- amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins, phosphorylation to form phosphoproteins, and a large number of chemical modifications (oxidation, deamidation, amidation, methylation, formylation, hydroxymethylation, guanidination, for example) as well as degraded, reduced, or crosslinked. The terms "polypeptide," "peptide" and "protein" include all unmodified and modified forms of the protein. A peptide would have a citrullinated residue or is part of the PAD enzyme.

[0041] "Detectable moiety" or a "label" refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include ³² P, ³⁵ S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavidin, digoxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target. The detectable moiety often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantify the amount of bound detectable moiety in a sample. Quantitation of the signal is achieved by, e.g., scintillation counting, densitometry, flow cytometry, or direct analysis by mass spectrometry of intact protein or peptides (one or more peptide can be assessed) that has a potential citrullinated residue or part of the PAD enzyme. Citrullinated Arg as part of a protein or peptide can be detected directly by MS or via chemical derivatization. Any capture reagent including but not limited to antibodies and aptamers may be used to detect cirullinated peptides described herein.

[0042] "Antibody" refers to a polypeptide ligand substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen). The recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad immunoglobulin variable region genes. Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab' and F(ab)'.sub.2 fragments. The term "antibody," as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CHI, CH2 and CH3, but does not include the heavy chain variable region.

[0043] By "binding assay" is meant a biochemical assay wherein the biomarkers are detected by binding to an agent, such as an antibody, through which the detection process is carried out. The detection process may involve radioactive or fluorescent labels, and the like. The assay may involve immobilization of the biomarker, or may take place in solution. Further, chemical binding to the citrullinated residue can occur directly. [0044] "Immunoassay" is an assay that uses an antibody to specifically bind an antigen (e.g., a marker). The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.

[0045] The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0046] Methods for detecting citrullination refer to the mass spectrometry (MS) based methods used to detect citrullinated peptides, polypeptides and proteins. The methods include but are not restricted to neutral loss of 1 Da when deimination occurs on Arg; neutral loss of isocyanic acid from unmodified citrulline and used this ion as a diagnostic marker for detecting protein citrullination; derivatization when chemical modification of 238 Da or 239 Da occurs on Cit residue (can be monitored at the peptide and protein level); enrichment of citrullinated peptides (or proteins) that is based on the specific reaction of glyoxal derivatives that is immobilized on beads/column/matrix reacts exclusively with the ureido group of the citrulline residue at low pH. As well, MS using a targeted method like multiple or selective reaction monitoring can be used to quantify the modified peptide directly. In some embodiments, a labeled (e.g. labeled with ¹⁵ N or chemically labeled with additional stable isotopes) peptide of known concentration is added to the sample and compared directly to the endogenous (unlabeled) corresponding peptide allowing for quantification (based on a ratio of the endogenous peptide to the isotopic labeled peptides or absolute concentration if the exact amount of the isotopic labeled peptide is known). [0047] The terms "subject", "patient" or "individual" generally refer to a human, although the methods of the invention are not limited to humans, and should be useful in other animals (e.g. birds, reptiles, amphibians, mammals), particularly in mammals, since albumin is homologous among species.

[0048] "Sample" is used herein in its broadest sense. A sample may comprise a bodily fluid including blood, serum, plasma, tears, aqueous and vitreous humor, spinal fluid; a soluble fraction of a cell or tissue preparation, or media in which cells were grown; or membrane isolated or extracted from a cell or tissue; polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; fragments and derivatives thereof. Subject samples usually comprise derivatives of blood products, including blood, plasma and serum.

[0049] The term "modulation of specific PAD isoforms" includes but is not limited to, increasing or decreasing the activity of endogenous PAD isoforms using gene therapy, siRNA, known inhibitors of PADs, or site-directed mutagenesis.

[0050] The term "peptide" as used herein refers to a polymer of amino acid residues typically ranging in length from 2 to about 30, or to about 40, or to about 50, or to about 60, or to about 70 residues. In certain embodiments the peptide ranges in length from about 2, 3, 4, 5, 7, 9, 10, or 11 residues to about 60, 50, 45, 40, 45, 30, 25, 20, or 15 residues. In certain embodiments the peptide ranges in length from about 8, 9, 10, 11, or 12 residues to about 15, 20 or 25 residues. In certain embodiments the amino acid residues comprising the peptide are "L-form" amino acid residues, however, it is recognized that in various embodiments, "D" amino acids can be incorporated into the peptide. Peptides also include amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In addition, the term applies to amino acids joined by a peptide linkage or by other, "modified linkages" (e.g., where the peptide bond is replaced by an a-ester, a f3-ester, a thioamide, phosphonamide, carbamate, hydroxylate, and the like (see, e.g., Spatola, (1983) Chern. Biochem. Amino Acids and Proteins 7: 267-357), where the amide is replaced with a saturated amine (see, e.g., Skiles et al., U.S. Pat. No. 4,496,542, which is incorporated herein by reference, and Kaltenbronn eta/., (1990) Pp. 969-970 in Proc. 11th American Peptide Symposium, ESCOM Science Publishers, The Netherlands, and the like)). [0051] Citrullination (also called deimination) is a post-translational protein modification catalyzed by PADs that involves conversion of the amino acid arginine to citrulline within proteins. The involvement of citrullinated proteins in the pathogenesis of a number of autoimmune diseases has been established. For example, protein citrullination has been directly linked to the generation of autoantibodies contributing to rheumatoid arthritis (RA). However, the exact circumstances and underlying purpose of PAD activation and consequent protein citrullination within cells are not fully understood. In addition, the production of autoantibodies targeting citrullinated proteins has been implicated in many other autoimmune diseases such as multiple sclerosis, psoriasis, sporadic Creutzfeld-Jakob disease, Parkinson's disease and Alzheimer's disease (AD). The knowledge of citrullinated proteins and citrullinated sites in protein sequences can provide invaluable information about the etiological importance and function of these citrullinated proteins. Provided herein are new biomarkers predictive of cardiovascular diseases and neurodegenerative diseases which include citrullinated proteins and peptides.

[0052] Citrullination is linked to autoantigen and autoantibody response which may be used for detection or measurement of ratios of autoantigen (unmodified/not-citrullinated) to autoantigen (modified/citrullinated) to detect heart disease (citrullinated autoantibody/autoantigens as biomarker). Further, detection of citrullinated auoantibody can be used as risk stratification factor. Risk of developing heart failure (FIF) and succumbing to myocardial infraction increases with age. Citrullination is known to increase with age and an increase in citrullination may be predictive of heart failure. Inhibition (eg. drug inhibition) of PAD enzyme responsible for citrullination may be a therapeutic target.

[0053] Protein citrullination plays a role in the generation of autoantibodies during the pathogenesis of specific neurodegenerative diseases such as Alzheimer's disease (AD). Since citrullination alters the overall charge distribution within a protein, potentially modifying tertiary structure, many citrullinated proteins in these PAD-expressing cells may be in a non- native conformation that is potentially immunogenic, especially if they are released from the cell and thus become accessible to immune surveillance.

[0054] Citrullination is linked to autoantigen and autoantibody response which may be used for detection or measurement of ratios of autoantigen (unmodified/not-citrullinated) to autoantigen (modified/citrullinated) to detect neurodegenerative disease (citrullinated autoantibody/autoantigens as biomarker). Citrullination is known to increase with age and an increase in citrullination may be predictive of neurodegenerative diseases. Inhibition (eg. drug inhibition) of PAD enzyme responsible for citrullination may be a therapeutic target.

Detection of peptidyl-citrulline - Comparison with other methods

[0055] Current research on citrullination focuses on its role in auto-immune diseases such as multiple sclerosis and rheumatoid arthritis (RA) [Baka Z, et al. Citrullination under physiological and pathological conditions. Joint Bone Spine 2012,79:431-436; De Ceuleneer M,et al. In vivo relevance of citrullinated proteins and the challenges in their detection. Proteomics 2012, 12:752-760]. In this context, the exact knowledge of citrullination sites in a protein can provide invaluable information about the etiological importance of these citrullinated proteins. However, few techniques exist that can accurately detect citrullination at the peptide level in a high-throughput format.

[0056] In general analysis of PTMs by MS is a difficult task and dependent on PTM profound effect on local peptide characteristics, like: (i) the mass shift in the peptide molecular weight induced by the PTM, (ii) the overall abundance of the modified peptide, (iii) the stability of the modification during MS and MS/MS analysis, and (iv) the effect of the modification on the peptide's ionization efficiency. Citrullination is complicated as citrullinated proteins / peptides are difficult to discern from their non-PTM forms because citrullination leads to an additional mass of nominally 0.9802 Dalton compared to regular arginine residues, which prone to false positives. The commonly applied shotgun type liquid chromatography tandem MS (LC-MS/MS) methods depend on automated database screening which in this case may cause misidentification of ¹³ C isotopes or misidentification of other modifications that lead to a similar mass increase (in particular the commonly occurring deamidation of glutamine or asparagine residues). Misidentification of 13C isotopes can be avoided by searching the database that has a small enough parent mass tolerance (<5 ppm), because ¹³ C leads to a slightly larger mass increase (1.0036 Da) than the conversion of arginine into citrulline (0.9802 Da) [De Ceuleneer M,et al. In vivo relevance of citrullinated proteins and the challenges in their detection. Proteomics 2012,12:752-760; Raij makers R, et al. Elevated levels of fibrinogen-derived endogenous citrullinated peptides in synovial fluid of rheumatoid arthritis patients. Arthritis Res Ther 2012, 14:R114]. Deamidation, however, cannot be discriminated from citrullination based on mass alone. Deamidation does not change the number of charged residues in the peptide (under the acidic conditions used for LC-MS), whereas the conversion of arginine to citrulline neutralizes a positive charge. Citrullination results in a loss of positive charge that causing a shift in the isoelectric point from 11.41 for arginine to 5.91 for citrulline. Therefore, citrullination increases the overall hydrophobicity of a peptide compared to the non-modified and/or deamidated peptide resulting in a shift in retention time, which is a proof for a peptide being citrullinated rather than deamidated [Raijmakers R, et al. Elevated levels of fibrinogen-derived endogenous citrullinated peptides in synovial fluid of rheumatoid arthritis patients. Arthritis Res Ther 2012, 14:R114.]. However, with the current methods it remains challenging to rapidly, consistently, reproducibly, accurately, and sensitively detect and quantify citrullinated peptides across multiple samples.

Advantages of SWATH MS over other strategies for citrullinated-targeted protein detection

[0057] The most commonly used mass spectrometry-based method for high-throughput protein citrullination analysis is data-dependent acquisition (DDA) [tahl DC, Set al. Data- controlled automation of liquid chromatography/tandem mass spectrometry analysis of peptide mixtures. J Am Soc Mass Spectrom 1996,7:532-540; Yates JR, 3rd, Eng TK, McCormack AL, Schieltz D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal Chem 1995,67: 1426-1436]. However, stochastic precursor selection and dependence on MSI ions for quantification impede comprehensive. To overcome this limitation a few years ago Aebersold's group came up with SWATH MS as a targeting method similar to multiple reaction monitoring (MRM). SWATH MS is a technique that combines data-independent acquisition (DIA) with targeted data extraction, which together gives quantitative accuracy, reproducibility, dynamic range, and extends the number of quantifiable peptides [Gillet LCI P TS, Rost H, Selevsek N, Reiter L, Bonner R, Aebersold R. . Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. . Mol Cell Proteomics 2012; Liu Y, et al. Mass spectrometric protein maps for biomarker discovery and clinical research. Expert Rev Mol Diagn 2013,13 :811-825]. Creation of highly citrullinated library improves the sensitivity and quantitative accuracy.

Cardiovascular Diseases

[0058] Provided herein are method for diagnosing and/or predicting cardiovascular diseases. The methods include obtaining a biological sample from a subject in need of diagnosing cardiovascular disease and detecting the amount of a citrullinated protein or a citrullinated peptide in the biological sample obtained from said subject. In some embodiments a change in the level of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increased risk of cardiovascular disease. In one embodiment, an increase in the levels of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increased risk of cardiovascular disease. In another embodiment, a decrease in the levels of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of increased risk of cardiovascular disease. In exemplary embodiments, change in cirullination levels of one or more proteins or peptides shown in Tables 1A, IB, 1C, ID and/or IE is indicative of increased risk of of cardiovascular diseases. In some embodiments, an increase in citrullination of the proteins shown in Table 1C, ID, IE and IF as indicated by "up" in Table 1C, ID, IE and IF is indicative of increased risk of cardiovascular disease. In some embodiments, a decrease in citrullination of the proteins shown in Table 1C, ID, IE and IF as indicated by "down" in Table 1C, ID, IE and IF is indicative of increased risk of cardiovascular disease. In some embodiments, change in citrullination refers to 'differentially present' citrullunation as defined herein

[0059] In some embodiments, a subject has ischemic heart disease (ISHD) or is at an increased risk of ISHD if the sample from the subject has an increase in citrullination in any one or more of the proteins in Table 1C, as indicated by 'up' in ischemic heart disease column (T). In some embodiments, the subject has or is at increased risk of ISHD if the sample from the subject has the presence of citrullination or an increase in citrullination of any one or more of the peptides in Table 1A. In some embodiments, the subject has or is at an increased risk of ISHD if the sample from the subject has the absence of citrullination or a decrease in citrullination of any one or more of the peptides in Table 1A. In some embodiments, a subject has or is at an increased risk of ISHD if the sample from the subject has an increase in cirullination in any one or more of ESI protein homolog, aconitate hydratase or citrullinated fragments thereof, glyceraldehyde-3 -phosphate dehydrogenase or citrullinated fragments thereof, delta- l-pyrroline-5-carboxylate dehydrogenase or citrullinated fragments thereof, vimentin or citrullinated fragments thereof, neuroblast differentiation-associated protein AHNAK or citrullinated fragments thereof, serum deprivation-response protein or citrullinated fragments thereof, enoyl-CoA hydratase or citrullinated fragments thereof, aconitate hydratase or citrullinated fragments thereof, or combinations thereof.

[0060] In some embodiments, a subject has idiopathic cardiomyopathy (IDCM) or is at increased risk of IDCM if the sample from the subject has an increase in citrullination in any one or more of the proteins in Table ID, as indicated by 'up' in IDCM column ('ID'). In some embodiments, the subject has or is at increased risk of IDCM if the sample from the subject has the presence of citrullination or an increase in citrullination of any one or more of the peptides in Table 1A. In some embodiments, the subject has or is at an increased risk of IDCM if the sample from the subject has the absence of citrullination or a decrease in citrullination of any one or more of the peptides in Table 1A. In some exemplary embodiments, a subject has or is at an increased risk of IDCM if the sample from the subject has an increase in citrullination in any one or more of ATP synthase subunit alpha or citrullinated fragments thereof, Myozenin-2 or citrullinated fragments thereof, Glyceraldehyde-3 -phosphate dehydrogenase or citrullinated fragments thereof, ESI protein homolog or citrullinated fragments thereof and/or combinations thereof.

[0061] In some embodiments, the subject has or is at increased risk of having cardiovascular disease (ISHD, IDCM or both ISHD and IDCM) if the sample from the subject has an increase in citrullination in any one or more of the proteins or peptides set forth in Tables 1 A- 1F. In some embodiments, the subject has or is at increased risk of having cardiovascular disease (ISHD, IDCM or both ISHD and IDCM) if the sample from the subject has an increase in citrullination in any one or more of ATP synthase subunit alpha or citrullinated fragments thereof, glyceraldehyde-3 -phosphate dehydrogenase or citrullinated fragments thereof, ESI protein homolog or citrullinated fragments thereof, delta- l-pyrroline-5- carboxylate dehydrogenase or citrullinated fragments thereof, enoyl-CoA hydratase or citrullinated fragments thereof, voltage-dependent anion-selective channel protein 1 or citrullinated fragments thereof, aconitate hydratase or citrullinated fragments thereof, pyruvate dehydrogenase El component subunit alpha, somatic form or citrullinated fragments thereof, vimentin or citrullinated fragments thereof, trifunctional enzyme subunit beta or citrullinated fragments thereof, myosin-binding protein C, cardiac-type or citrullinated fragments thereof, chloride intracellular channel protein 4 or citrullinated fragments thereof, heat shock protein HSP 90-alpha or citrullinated fragments thereof, serum deprivation- response protein or citrullinated fragments thereof, alcohol dehydrogenase IB or citrullinated fragments thereof, succinate dehydrogenase [ubiquinone] iron-sulfur subunit or citrullinated fragments thereof, dual specificity protein phosphatase 3 or citrullinated fragments thereof or combinations thereof (Table IE). In some embodiments, the subject has or is at increased risk of having cardiovascular disease (ISHD, IDCM or both ISHD and IDCM) if the sample from the subject has a decrease in citrullination in any one or more of Troponin I, Alpha- crystalline B chain, ATP-synthase-coupling factor 6, beta-enolase, adenylate kinase isoenzyne I or combination thereof (Table IF). In some embodiments, change in citrullination refers to 'differentially present' citrullination as defined herein.

[0062] Also provided herein is a method comprising obtaining a biological sample from a subject in need of diagnosing cardiovascular disease, using a peptide to assay the biological sample to detect the presence or absence of an antibody capable of binding specifically to the peptide, determining the presence of cardiovascular disease when the presence of the antibody capable of binding specifically to the peptide is detected and directing the subject to treatment of cardiovascular disease when the presence of the antibody capable of binding specifically to the peptide is detected. In various embodiments, presence of the antibody capable of binding specifically to the peptide is indicative of any one or more of ISHD, IDCM, heart failure or a combination thereof. In some embodiments, the peptide is a fragment of Troponin I, Vimentin, Myosin-7 or a combination thereof. In various embodiments, the peptide is citrullinated. In exemplary embodiments, the peptide comprises, consists of or consists essentially of the amino acid sequence NIDALSGMEGRK (SEQ ID NO: 18), PRSFMPNLVPPK (SEQ ID NO: 20), ESLDLRAHLK (SEQ ID NO: 19), AEETQRSVNDLTSQRAK (SEQ ID NO: 24) or FADLSEAANRNNDALRQAK (SEQ ID NO: 22). In exemplary embodiments, the peptide sequence of the citrullinated peptides in the sample from the subject is at least 95%, 90%, 80%>, 70% or 60% identical to peptides set forth in any of Table 1A-1F. In exemplary embodiments, the peptide sequence of the citrullinated peptides in the sample from the subject is at least 95%, 90%, 80%, 70% or 60% identical to NIDALSGMEGRK (SEQ ID NO: 18), PRSFMPNLVPPK (SEQ ID NO: 20), ESLDLRAHLK (SEQ ID NO: 19), AEETQRSVNDLTSQRAK (SEQ ID NO: 24) or FADLSEAANRNNDALRQAK (SEQ ID NO: 22). In various embodiments, the length of the peptide is optimized to bind the antibody in the sample. In various embodiments, citrullinated peptides are detected using mass spectrometry, high resolution mass spectrometry, tandem mass spectrometry, binding assay, immunoassay, SDS page electrophoresis, Western blot analysis, conformation on mass spectrometry, including SWATH.

[0063] In various embodiments, the methods further comprise prescribing treatment to the subject diagnosed with cardiovascular disease. In various embodiments, treatments include but are not limited to any one or more of therapeutic lifestyle changes, therapeutic agents, surgical treatments or a combination thereof. In exemplary embodiments, lifestyle changes include any one or more of healthy diet, physical activity, weight management, stress management, not smoking or a combination thereof. In exemplary embodiments, therapeutic agents include any one or more of Angiotensin-converting enzyme (ACE) inhibitors, Angiotensin II receptor blockers (ARBs), Beta-blockers, Aldosterone antagonists, Cardiac glycosides, Diuretics, Vasodilators, Antiarrhythmics, Human B-type natriuretic peptide, Inotropic agent, Anticoagulants, inhibitors of peptidyl arginine deiminase (PAD) enzymes or a combination thereof. In exemplary embodiments, surgical treatments include any one or more of use of left ventricular assist devices, cardiac resynchronization therapy (biventricular pacing), automatic implantable cardioverter-defibrillators, ventricular restoration surgery, heart transplantation, angioplasty, stents, atherectomy, radiation therapy, coronary artery bypass grafting or a combination thereof. Appropriate therapies will be apparent to one skilled in the art.

[0064] In various embodiments of the processes, assays and methods described herein, the reference value is based on the change in the level of citrullinated proteins or citrullinated peptides, as set forth in, for example, Tables 1A, IB, 1C, ID, IE and/or IF. In one embodiment, the change in the level of citrullinated proteins or citrullinated peptides is in a cardiomyocyte. In another embodiment, the change in the level of citrullinated proteins or citrullinated peptides is in a non-cardiac cell. In an additional embodiment, the change in the level of citrullinated proteins or citrullinated peptidesis in any cell. In an additional embodiment, the change in the level of citrullinated proteins or citrullinated peptidesis in any bodily fluid. In some embodiments, the reference value is the mean or median change in level of citrullinated proteins or citrullinated peptides in a population of subjects that do not have cardiovascular disease. In other embodiments, the reference value is the mean or median change in level of citrullinated proteins or citrullinated peptides in a population of subjects that have cardiovascular diseases but have undergone treatment and show no or reduced expression of citrullinated proteins or citrullinated peptides. In additional embodiments, the reference value is the change in expression level of citrullinated proteins or citrullinated peptides in a sample obtained from the subject from a different (for example, an earlier) time point, such as during diagnosis, before treatment, after treatment or a combination thereof. In some embodiments, cardiovascular disease is any one or more of ISHD, IDCM, heart failure or a combination thereof. In some embodiments, change in citrullination refers to 'differentially present' citrullination as defined herein. [0065] In various embodiments, the change in the level of citrullinated proteins or citrullinated peptides in the subject having or suspected of having cardiovascular disease compared to the reference value is increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In various embodiments, the change in the level of citrullinated proteins or citrullinated peptides in the subject having or suspected of having cardiovascular disease compared to the reference value is increased by at least or about 1- fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40- fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold or a combination thereof.

[0066] In various embodiments, the change in the level of citrullinated proteins or citrullinated peptides in the subject having or suspected of having cardiovascular disease compared to the reference value is decreased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In various embodiments, the change in the level of citrullinated proteins or citrullinated peptides in the subject having or suspected of having cardiovascular disease compared to the reference value is decreased by at least or about 1- fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40- fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold or a combination thereof.

Neurodegenerative Diseases

[0067] Provided herein are methods for diagnosing and/or predicting chronic neurodegenerative diseases and/or acquired brain injury and/or acute brain disease. The methods include obtaining a biological sample from a subject in need of a diagnosis and detecting citrullinated protein or citrullinated peptide in the biological sample obtained from said subject. In some embodiments, presence of citrullinated proteins or citrullinated peptides is indicative of neurodegenerative diseases. In some embodiments a change in the level of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of neurodegenerative disease and/or acquired brain injury and/or acute brain disease. In one embodiment, an increase in the levels of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of neurodegenerative disease and/or acquired brain injury and/or acute brain disease. In another embodiment, a decrease in the levels of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of neurodegenerative diseaseand/or acquired brain injury and/or acute brain disease. In exemplary embodiments, a change in the levels of one or more citrullinated peptides or citrullinated proteins shown in Tables 2-10 is indicative of neurodegenerative diseases. In exemplary embodiments, a change in the amount of citrullination of one or more peptides or proteins shown in Tables 2-10 is indicative of neurodegenerative diseases. In some embodiments, the change in the level is an increase in the level of one or more citrullinated peptides or citrullinated proteins shown in Tables 2-10 which is indicative of chronic neurodegenerative disease. In some embodiments, the change in the level is a decrease in the level of one or more citrullinated peptides or citrullinated proteins shown in Tables 2-10 which is indicative of chronic neurodegenerative disease. In some embodiments, change in citrullination refers to 'differentially present' citrullination as defined herein.

[0068] In exemplary embodiments, examples of neurodegenerative diseases include but are not limited to Alzheimer disease, Parkinson disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington disease, Friedreich's ataxia, spinal muscular atrophy, prion diseases, multiple sclerosis, stroke, degenerative nerve diseases, encephalitis, Motor neurone diseases (MND, Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA) or Creutzfeld-Jacob disease.

[0069] In some embodiments, the biological sample is any one or more of blood, plasma, serum, urine or tissue (tissue biopsy).

[0070] In various embodiments, the level of the citrullinated protein or citrullinated peptide is detected using mass spectrometry, high resolution mass spectrometry, tandem mass spectrometry, binding assay, immunoassay, antibody binding or immunohistochemistry.

[0071] Provided herein is a method for diagnosing ALS in a subject in need thereof. The method includes obtaining a biological sample from a subject in need of a diagnosis and detecting citrullinated protein or citrullinated peptide in the biological sample obtained from the said subject. In some embodiments, presence of citrullinated proteins or citrullinated peptides is indicative of ALS. In some embodiments a change in the level of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of ALS. In one embodiment, an increase in the levels of citrullination of protein or citrullination of peptide relative to a reference value is indicative of ALS. In one embodiment, the subject has ALS or is at increased risk of having ALS if the sample from the subject has an increase in citrullination in any one or more of the proteins in Table 2, Table 3 or Table 4. In another embodiment, the subject has ALS or is at increased risk of having ALS if the sample from the subject has an increase in citrullination in any one or more of the peptides PSGVQMDPCCRALYDFEPE EGELGFK (SEQ ID NO: 306), QYMRRSTCTINYSK (SEQ ID NO: 307), LILIARNK (SEQ ID NO: 308), VI VVWVGTNNHENTAEEV AGGIE AIVQLINTRQPQAK (SEQ ID NO: 309), EVDVGLAADVGTLQRLPK (SEQ ID NO: 310), HEEAPGHRPTTNPNASK (SEQ ID NO: 311), GETPVNSTMSIGQARK (SEQ ID NO: 312), VRIQTQPGYANTLRDAAPK (SEQ ID NO: 313), NVGCLQEALQL AT SF AQLRLGD VK (SEQ ID NO: 314) or combinations thereof. In exemplary embodiments, the citrullination sites in peptides having the sequence set forth in SEQ ID Nos. 306-314 are shown in Table 2. In a further embodiment, the subject has ALS or is at increased risk of having ALS if the sample from the subject has an increase in citrullination in any one or more of the peptides having the sequence set forth in any one or more of SEQ ID NO. : 315, SEQ ID NO. : 316, SEQ ID NO. : 317, SEQ ID NO.: 318, SEQ ID NO. : 319, SEQ ID NO.: 320, SEQ ID NO. : 321, SEQ ID NO.: 322, SEQ ID NO. : 323, SEQ ID NO. : 324, SEQ ID NO. : 325, SEQ ID NO. : 326, SEQ ID NO.: 327, SEQ ID NO.: 328, SEQ ID NO.: 329, SEQ ID NO.: 330, SEQ ID NO.: 331, SEQ ID NO.: 332, SEQ ID NO. : 333, SEQ ID NO.: 334, SEQ ID NO.: 335, SEQ ID NO.: 336, SEQ ID NO. : 337, SEQ ID NO. : 338, or combinations thereof. In exemplary embodiments, the citrullination sites in peptides having the sequence set forth in SEQ ID Nos. 315-338 are shown in Table 3. In exemplary embodiments, each of the citrullinatedpeptide sequences in the sample obtained from the subhect is at least 100% 95%, 90%, 80%, 70% or 60% identical to the sequences set forth in any of SEQ ID Nos. 306-338. In some embodiments, the subject has ALS or is at increased risk of having ALS if the sample from the subject has an increase in citrullination in any one or more of the peptides having the sequence set forth in any one or more of SEQ ID Nos. 339-474. In exemplary embodiments, each of the peptide sequence in the sample obtained from the subject is at least 100%, 95%, 90%, 80%, 70% or 60% identical to the sequences set forth in any of SEQ ID Nos. 339-474. In some embodiments, change in citrullination refers to 'differentially present' citrullination as defined herein.

[0072] Provided herein is a method for diagnosing chronic neurodegenerative disease in a subject in need thereof. The method includes obtaining a biological sample from a subject in need of a diagnosis and detecting citrullinated protein or citrullinated peptide in the biological sample obtained from the said subject. In some embodiments, presence of citrullinated proteins or citrullinated peptides is indicative of chronic neurodegenerative disease. In some embodiments a change in the level of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of chronic neurodegenerative disease. In one embodiment, an increase in the levels of citrullination of protein or citrullination of peptide relative to a reference value is indicative of chronic neurodegenerative disease. In one embodiment, a decrease in the levels of citrullination of protein or citrullination of peptide relative to a reference value is indicative of chronic neurodegenerative disease. In one embodiment, the subject has chronic neurodegenerative disease or is at increased risk of having chronic neurodegenerative disease if the sample from the subject has a change in in citrullination in any one or more of the proteins in Table 5A. In some embodiments, the citrullinated peptides are brain specific. In some embodiments, the citrullinated peptides are not brain specific. In a further embodiment, the subject has chronic neurodegenerative disease or is at increased risk of having chronic neurodegenerative disease if the sample from the subject has a change in citrullination in any one or more of the peptides having the sequence set forth in any one or more of SEQ ID NO. : 355, SEQ ID NO.: 402, SEQ ID NO.: 1, SEQ ID NO.: 348, SEQ ID NO.: 363, SEQ ID NO.: 428, SEQ ID NO.: 373, SEQ ID NO.: 315, SEQ ID NO. : 316, SEQ ID NO. : 340, SEQ ID NO. : 377, SEQ ID NO. : 331, SEQ ID NO. : 332, SEQ ID NO.: 325, SEQ ID NO. : 375, SEQ ID NO.: 429, SEQ ID NO. : 414, SEQ ID NO. : 313, SEQ ID NO. : 366, SEQ ID NO. : 360, SEQ ID NO.: 423, SEQ ID NO.: 405, SEQ ID NO.: 439, SEQ ID NO.: 321, SEQ ID NO.: 475, SEQ ID NO.: 476, SEQ ID NO. : 477, SEQ ID NO.: 478, SEQ ID NO.: 479, SEQ ID NO.: 480, SEQ ID NO. : 481, SEQ ID NO. : 482, SEQ ID NO.: 483, SEQ ID NO. : 484, SEQ ID NO.: 485, SEQ ID NO. : 486, SEQ ID NO. : 487, SEQ ID NO. : 488, SEQ ID NO. : 489, SEQ ID NO.: 490, SEQ ID NO.: 491, SEQ ID NO.: 492, SEQ ID NO.: 493, SEQ ID NO.: 494, SEQ ID NO.: 495, SEQ ID NO. : 496, SEQ ID NO.: 497, SEQ ID NO. : 498, SEQ ID NO. : 499, SEQ ID NO.: 500, or combinations thereof (Table 5A). In exemplary embodiments, the citrullination sites in peptides having the sequence set forth in SEQ ID Nos. above are shown in Table 5A. In exemplary embodiments, each of the citrullinated peptide sequences in the sample from the subject is at least 95%, 90%, 80%, 70% or 60% identical to the sequences set forth Table 5 A. In exemplary embodiments, peptides that may be used for diagnosing chronic neurodegenerative disease are brain specific and comprises, consists of or consists essentially of any one or more of the sequences shown in Table 5A. In exemplary embodiments, peptides that may be used for diagnosing chronic neurodegenerative disease are not brain specific and comprises, consists of or consists essentially of sequences set forth in SEQ ID Nos. 501, 502, 503, 506 or combinations thereof (Table 5B). In exemplary embodiments, chronic neurodegenerative disease include buy are not limited to any one or more of Alzheimer's disease (AD) and other dementias, Parkinson's disease (PD) and PD-related disorders, Prion disease, Motor neurone diseases (MND), Huntington's Disease (HD), Spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA) or combinations thereof. In some embodiments, change in citrullination refers to 'differentially present' citrullination as defined herein.

[0073] Provided herein is a method for diagnosing acquired brain injury in a subject in need thereof. The method includes obtaining a biological sample from a subject in need of a diagnosis and detecting citrullinated protein or citrullinated peptide in the biological sample obtained from the said subject. In some embodiments, presence of citrullinated proteins or citrullinated peptides is indicative of acquired brain injury. In some embodiments a change in the level of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of acquired brain injury. In one embodiment, an increase in the levels of citrullination of protein or citrullination of peptide relative to a reference value is indicative of acquired brain injury. In one embodiment, a decrease in the levels of citrullination of protein or citrullination of peptide relative to a reference value is indicative of acquired brain injury. In a further embodiment, the subject has acquired brain injury or is at increased risk of having acquired brain injury if the sample from the subject has a change in citrullination in any one or more of the peptides having the sequence set forth in any one or more of SEQ ID NO. : 125, SEQ ID NO.: 265, SEQ ID NO. : 285, SEQ ID NO.: 505, SEQ ID NO. : 506, SEQ ID NO. : 507, SEQ ID NO. : 508, SEQ ID NO. : 509, SEQ ID NO.: 510, SEQ ID NO.: 511, SEQ ID NO.: 512, SEQ ID NO.: 513, SEQ ID NO.: 514, SEQ ID NO.: 515, SEQ ID NO. : 516, SEQ ID NO.: 517, SEQ ID NO.: 518, SEQ ID NO.: 519, SEQ ID NO. : 520, SEQ ID NO. : 521, SEQ ID NO.: 522, SEQ ID NO. : 523, SEQ ID NO.: 524, SEQ ID NO. : 525, SEQ ID NO. : 526, SEQ ID NO. : 527, SEQ ID NO. : 528, SEQ ID NO.: 529, SEQ ID NO.: 530, SEQ ID NO.: 531, SEQ ID NO.: 532, SEQ ID NO.: 533, SEQ ID NO.: 534, SEQ ID NO. : 535, SEQ ID NO.: 536, SEQ ID NO.: 537, SEQ ID NO.: 538, SEQ ID NO. : 539, SEQ ID NO. : 540, SEQ ID NO.: 541, SEQ ID NO. : 542, SEQ ID NO.: 543, SEQ ID NO. : 544, SEQ ID NO.: 545, SEQ ID NO.: 546, SEQ ID NO.: 547 or combinations thereof. In a further embodiment, the subject has acquired brain injury or is at increased risk of having acquired brain injury if the sample from the subject has a change in citrullination in any one or more of the peptides having the sequence set forth in Tables 6A and/or 6B. In exemplary embodiments, each of the citrullinated peptide sequence in the sample obtained from the subject is at least 95%, 90%, 80%, 70% or 60% identical to the sequences set forth in any of Tables 6A and/or 6B. In exemplary embodiments, acquired brain injury includes but is not limited to traumatic brain injuries (TBI's), strokes, brain illness, tumor, hemorrhage, and any other kind of brain injury acquired after birth. In some embodiments, change in citrullination refers to 'differentially present' citrullination as defined herein.

[0074] Provided herein is a method for diagnosing chronic brain disease in a subject in need thereof. The method includes obtaining a biological sample from a subject in need of a diagnosis and detecting citrullinated protein or citrullinated peptide in the biological sample obtained from the said subject. In some embodiments, presence of citrullinated proteins or citrullinated peptides is indicative of chronic brain disease. In some embodiments a change in the level of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of chronic brain disease. In one embodiment, an increase in the levels of citrullination of protein or citrullination of peptide relative to a reference value is indicative of chronic brain disease. In one embodiment, a decrease in the levels of citrullination of protein or citrullination of peptide relative to a reference value is indicative of chronic brain disease. In one embodiment, the subject has chronic brain disease or is at increased risk of having chronic brain disease if the sample from the subject has a change in citrullination in any one or more of the secreted proteins in Table 7. In one embodiment, the subject has chronic brain disease or is at increased risk of having chronic brain disease if the sample from the subject has a change in citrullination in any one or more of the non-secreted proteins in Table 8. In a further embodiment, the subject has chronic brain disease or is at increased risk of having chronic brain disease if the sample from the subject has a change in citrullination in any one or more of secreted proteins selected from Syntaxin-binding protein 1 or citrullinated fragments thereof, Histone H4 or citrullinated fragments thereof, Cytoplasmic dynein 1 heavy chain 1 or citrullinated fragments thereof, Dynamin-1 or citrullinated fragments thereof, Gamma-enolase or citrullinated fragments thereof, Endoplasmin precursor or citrullinated fragments thereof, Heat shock 70 kDa protein 12A or citrullinated fragments thereof, Serine/threonine-protein phosphatase PP1 -alpha catalytic subunit or citrullinated fragments thereof, Calreticulin precursor or citrullinated fragments thereof, Coronin-IA or citrullinated fragments thereof, Electron transfer flavoprotein subunit beta or citrullinated fragments thereof, Hemoglobin subunit beta or citrullinated fragments thereof, Mitogen-activated protein kinase 1 or citrullinated fragments thereof, Mitogen-activated protein kinase 3 or citrullinated fragments thereof, 26 S proteasome non-ATPase regulatory subunit 12 or citrullinated fragments thereof, 60S ribosomal protein L4 or citrullinated fragments thereof, 60S acidic ribosomal protein P2 or citrullinated fragments thereof, Septin-7 or citrullinated fragments thereof, Alanine-tRNA ligase, cytoplasmic or citrullinated fragments thereof or combinations thereof. In a further embodiment, the subject has chronic brain disease or is at increased risk of having chronic brain disease if the sample from the subject has a change in citrullination in any one or more of non-secreted proteins selected from Synapsin-1 or citrullinated fragments thereof, Aconitate hydratase or citrullinated fragments thereof, Complexin-1 or citrullinated fragments thereof, Complexin-2 or citrullinated fragments thereof, Band 4.1 -like protein 3 or citrullinated fragments thereof, Synapsin-2 or citrullinated fragments thereof, AP2-associated protein kinase 1 or citrullinated fragments thereof, Alpha- adducin or citrullinated fragments thereof, AP-2 complex subunit beta or citrullinated fragments thereof, Serine/threonine-protein kinase DCLK1 or citrullinated fragments thereof, Glutaminase kidney isoform or citrullinated fragments thereof, Isocitrate dehydrogenase [NAD] subunit alpha or citrullinated fragments thereof, Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform or citrullinated fragments thereof or combinations thereof. In some embodiments, change in citrullination refers to 'differentially present' citrullination as defined herein.

[0075] Provided herein is a method for diagnosing acute brain disease in a subject in need thereof. The method includes obtaining a biological sample from a subject in need of a diagnosis and detecting citrullinated protein or citrullinated peptide in the biological sample obtained from the said subject. In some embodiments, presence of citrullinated proteins or citrullinated peptides is indicative of acute brain disease. In some embodiments a change in the level of the citrullinated protein or the citrullinated peptide relative to a reference value is indicative of acute brain disease. In one embodiment, an increase in the levels of citrullination of protein or citrullination of peptide relative to a reference value is indicative of acute brain disease. In one embodiment, a decrease in the levels of citrullination of protein or citrullination of peptide relative to a reference value is indicative of acute brain disease. In one embodiment, the subject has acute brain disease or is at increased risk of having acute brain disease if the sample from the subject has a change in citrullination in any one or more of the secreted proteins in Table 9. In one embodiment, the subject has acute brain disease or is at increased risk of having acute brain disease if the sample from the subject has a change in citrullination in any one or more of the non-secreted proteins in Table 10. In a further embodiment, the subject has acute brain disease or is at increased risk of having acute brain disease if the sample from the subject has a change in citrullination in any one or more of secreted proteins selected from Cullin-associated EDD8-dissociated protein 1 or citrullinated fragments thereof, Peroxiredoxin-5 or citrullinated fragments thereof or combinations thereof. In a further embodiment, the subject has acute brain disease or is at increased risk of having acute brain disease if the sample from the subject has an increase in citrullination Guanine nucleotide-binding protein G(I)/G(S)/G(0) subunit gamma-3 precursor or citrullinated fragments thereof. In some embodiments, change in citrullination refers to 'differentially present' citrullination as defined herein.

[0076] Also provided herein are methods for treating neurodegenerative diseases in a subject in need thereof. The methods include diagnosing a neurodegenerative disease by the methods set forth herein and administering an effective amount of a therapeutic agent to the subject diagnosed with the neurodegenerative disease so as to treat neurodegenerative disease in the subject. In exemplary embodiments, examples of neurodegenerative diseases include but are not limited to Alzheimer disease, Parkinson disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington disease, Friedreich's ataxia, spinal muscular atrophy, prion diseases, multiple sclerosis, stroke, degenerative nerve diseases, encephalitis or Creutzfeld- Jacob disease. In various embodiments, treatments include but are not limited to any one or more of therapeutic lifestyle changes, therapeutic agents, surgical treatments or a combination thereof. In exemplary embodiments, lifestyle changes include any one or more of healthy diet, physical activity, weight management, stress management, not smoking or a combination thereof.

[0077] In exemplary embodiments, therapeutic agents for Alzheimer's disease include but are not limited to cholinesterase inhibitors (such as Donepezil, Rivastigmine, Galantamine), memantine (Namenda), Vitamin E, alternative treatments (for example, Caprylic acid and coconut oil, Concerns, Coenzyme Q10, Coral calcium, Ginkgo biloba, Huperzine A, Omega- 3 fatty acids, Phosphatidylserine, Tramiprosate), or combinations thereof. Appropriate therapies and dosages will be apparent to one skilled in the art.

[0078] In exemplary embodiments, therapeutic agents for Parkinson's disease include but are not limited to carbidopa/levodopa, dopamine agonists, monoamine oxidase inhibitors, anticholinergics, surgery, lifestyle changes, alternative treatments (for example, Antioxidants Vitamin C and E, the Mediterranean Diet, Calcium and Vitamin D, Coenzyme Q10, Folate (Folic Acid), Ginger (Zingiber Officinalis), Gingko Biloba, Green Tea Polyphenols (GTPs), Milk Thistle (Silybum Marianum), St. John's Wort (Hypericum Perforatum), Vitamin B12) or combinations thereof. Appropriate therapies and dosages will be apparent to one skilled in the art.

[0079] In exemplary embodiments, treatments for Huntinton's disease include therapeutic agents and therapies including psychotherapy, speech therapy, physical therapy, occupation therapy or combinations thereof. Exemplary, therapeutic agents for Huntington's disease include but are not limited to Tetrabenazine (Xenazine), Antipsychotic drugs, (such as haloperidol (Haldol)), amantadine, levetiracetam (Keppra), clonazepam (Klonopin) or combinations thereof. Appropriate therapies and dosages will be apparent to one skilled in the art.

[0080] In exemplary embodiments, therapeutic agents for ataxia include but are not limited to riluzole, antiglutaminergic medication, nicotine receptor agonists, serotonergic therapy, GABAergic therapy, cholinergic therapy, channel stabilizing treatments (such as carbonic anhydrase inhibitors), insulin-like growth factors, supplements (such as Vitamin E) or combinations thereof. Appropriate therapies and dosages will be apparent to one skilled in the art.

[0081] In exemplary embodiments, treatments for spinal muscular atrophy include proper nutrition, therapeutic agents and therapies including psychotherapy, speech therapy, physical therapy, occupation therapy or combinations thereof. Therapeutic agents for spinal muscular atrophy include but are not limited to muscle relaxants such as baclofen, tizanidine, and the benzodiazepines to reduce spasticity, Botulinum toxin to treat jaw spasms or drooling, amitriptyline, glycopyolate, and atropine or by botulinum injections into the salivary glands to treat excessive saliva, antidepressants to treat depression. Appropriate therapies and dosages will be apparent to one skilled in the art.

[0082] In exemplary embodiments, therapeutic agents for prion disease include but are not limited to quinacrine, pentosan polysulfate (PPS), tetracyclic compounds or combinations thereof.

[0083] In various embodiments of the processes, assays and methods described herein, the reference value is based on the change in the level of citrullinated proteins or citrullinated peptides as set forth in, for example Tables 2-10. In one embodiment, the change in the level of citrullinated proteins or citrullinated peptides is in a neuronal cell. In another embodiment, the change in the level of citrullinated proteins or citrullinated peptides is in a non-neuronal cell. In an additional embodiment, the change in the level of citrullinated proteins or citrullinated peptides is in any cell. In an additional embodiment, the change in the level of citrullinated proteins or citrullinated peptides is in any bodily fluid. In some embodiments, the reference value is the mean or median expression level of citrullinated proteins or citrullinated peptides in a population of subjects that do not have neurodegenerative disease. In other embodiments, the reference value is the mean or median expression level of citrullinated proteins or citrullinated peptides in a population of subjects that have neurodegenerative diseases but have undergone treatment and show no or reduced expression of citrullinated proteins or citrullinated peptides. In additional embodiments, the reference value is the expression level of citrullinated proteins or citrullinated peptides in a sample obtained from the subject from a different (for example, an earlier) time point, such as during diagnosis of neurodegenerative disease, before treatment of neurodegenerative disease, after treatment of neurodegenerative diseaseor a combination thereof. In exemplary embodiments, neurodegenerative diseases include but are not limited to ALS, Alzheimer's disease, brain stroke, chronic brain disease, acute brain disease or combinations thereof. In some embodiments, change in citrullination refers to 'differentially present' citrullination as defined herein.

[0084] In various embodiments, the expression level of citrullinated proteins or citrullinated peptides in the subject having or suspected of having neurodegenerative disease compared to the reference value is increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%), 90% or 100%). In various embodiments, the expression level of citrullinated proteins or citrullinated peptides in the subject having or suspected of having neurodegenerative disease compared to the reference value is increased by at least or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold or a combination thereof. In exepmlary embodiments, neurodegenerative diseases include but are not limited to ALS, Alzheimer's disease, brain stroke, chronic brain disease, acute brain disease or combinations thereof.

[0085] In various embodiments, the expression level of citrullinated proteins or citrullinated peptides in the subject having or suspected of having neurodegenerative disease compared to the reference value is decreased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%), 90%) or 100%). In various embodiments, the expression level of citrullinated proteins or citrullinated peptides in the subject having or suspected of having neurodegenerative disease compared to the reference value is decreased by at least or about 1-fold, 2-fold, 3 -fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold or a combination thereof. In exepmlary embodiments, neurodegenerative diseases include but are not limited to ALS, Alzheimer's disease, brain stroke, chronic brain disease, acute brain disease or combinations thereof.

Example 1: Experimental Methods

Reagents and materials

[0086] The following reagents were obtained: rabbit skeletal muscle PAD cocktail (PAD) (SignalChem); PAD2 (Sigma), heavy meromyosin (HMM), tropomyosin (TM) (Sigma); F- actin (Cytoskeleton.Inc), cardiac troponin (Tnl) (Abeam), anti-modified citrulline antibody (Millipore); sequencing grade Lys-C protease (WAKO), and protease inhibitor cocktail (Roche).

Human heart tissue

[0087] Left ventricular tissue samples were obtained from Cris Dos Remedios, University of Sydney, Australia after informed consent and with approval of the local Ethical Committee. The samples were acquired during heart transplantation surgery, from patients with FIF (Ischemic Heart Disease (ISHD) and Idiopathic Cardiomyopathy (IDCM), n=10 each) and non-failing donor hearts (n=10) as previously described [Zhang P et al.. Multiple reaction monitoring to identify site-specific troponin I phosphorylated residues in the failing human heart. Circulation 2012, 126: 1828-1837].

Mouse heart tissue and neonatal myocytes

[0088] Male C57BL/6 mice (n=3) (5 days old neonatal mice, Jackson Laboratories) were obtained. Animal study was approved by The Johns Hopkins University Animal Care and Use Committee and followed established NIH guidelines. Briefly, Primary cultured ventricular myocytes were isolated from neonatal mice. Collagenase-digested isolated myocytes were incubated in buffer with increasing concentrations of Ca ²⁺ , achieving a final concentration of 1.2 mM Ca ²⁺ as in the MEM culture media. Cells were seeded at 25,000 rod- shaped myocytes/ml on 6-well plates or 60-mm dishes coated with laminin. After 1 hour incubation in 37°C, 5% C0 ₂ , the culture media was replaced to remove unattached cells. Protein extraction and SWATH MS

[0089] Hearts were fractionated into myofilament- and cytosolic-enriched fractions using the IN Sequence protocol [Kane LA et al. Subfractionation of heart tissue: the "in sequence" myofilament protein extraction of myocardial tissue. Methods Mol Biol 2007, 357:87-90]. Protein extraction and generation of LysC peptides from subfractions was performed using a filter-aided sample preparation (FASP) protocol [24]. When needed recombinant proteins or the In Sequence fractions were incubated with PAD's cocktail at a ratio of 1 :20 for 2 hr. at 37°C in lOOmM Tris, pH 7.6, 5mM DTT, lOmM CaCl ₂ . The reaction was stopped by addition of 5 mM EDTA prior to digestion. A TripleTOF 6600 mass spectrometer (Sciex) was used for both data dependent acquisition to build peptide spectral ion library and SWATH-MS (data independent acquisition) for each individual sample analysis. The raw data was searched with ProteinPilot™ Software 5.0 to create a spectral ion library. Individual SWATH-MS runs were matched against the spectral library created in the presence or absence of PAD (plus and minus PAD) for both the myofilament- and cytosolic-enriched protein fractions (see Data Supplement).

Preparation of citrullinated samples

[0090] Recombinant proteins and the fractions obtained from IN Sequence were incubated with PAD2 at a ratio of 1 :20 for 2 hr. at 37°C in working buffer (lOOmM Tris, pH 7.6, 5mM DTT, lOmM CaCl ₂ ). The reaction was stopped by addition of 5 mM EDTA.

Statistical validation peptides, proteins and citrullination residues: Acceptance Criteria.

[0091] Bioinformatics Analysis was performed with the workflow described in data supplement. The peptide normalization used in this study was based on the iRT peptide retention time [Escher C, et al. Using iRT, a normalized retention time for more targeted measurement of peptides. Proteomics 2012, 12: 1111-1121] and normalized values were used for downstream analysis. Ensemble protein ID accession numbers were mapped back to their associated encoding Ensemble gene entries. Data analysis and mining were performed using iProXpress (http://proteininformationresource.org/iproxpress2) [Natale DA et al. Protein Ontology: a controlled structured network of protein entities. Nucleic Acids Res 2014, 42:D415-421] and Cytoscape [Saito R et al. A travel guide to Cytoscape plugins. Nat Methods 2012, 9: 1069-1076]. The Kruskal-Wallis test (non-parametric one way ANOVA) for each peptide was used to calculate p-values. The significance of the biochemical changes was determined by performing a t-test (p<0.05) on the differences for all paired data. Unless otherwise stated all biochemical assays were replicated three times.

SDS-PAGE immunoblot for citrullination

[0092] A 1 :2000 diluted of the anti-citrulline (Modified) antibody was used for the 1 DE western blot (see Data Supplement).

Two-Dimensional Gel Electrophoresis (2DE)

[0093] The independent verification of proteomics data was performed with fluorescence 2DE gel electrophoresis (2D-DIGE, pi range and SDS PAGE range) as reported previously [Matt P et al. Biomarker discovery: proteome fractionation and separation in biological samples. Physiol Genomics 2008, 33 : 12-17]. The treatment of sample with PAD2 enzyme, which removes a guanidino group from specific arginine residues within the modified protein, can be used to identify citrullinated proteins based on the change in charge of the protein after treatment (see Data Supplement).

Membrane-permeabilized myocytes

[0094] Left ventricular tissue from C57BL/6 mice was flash frozen in liquid nitrogen and stored at -80 °C. For analysis, tissue was homogenized in the presence of 0.3% Triton X-100, and protease and phosphatase inhibitors, as described [Kirk JA et al. Cardiac resynchronization sensitizes the sarcomere to calcium by reactivating GSK-3beta. J Clin Invest. 2014, 1 : 129-38]. Myocytes were washed without Triton X-100 to remove the detergent, and resuspended in isolation buffer. PAD2 was activated in 10 mM Ca ²⁺ and 50 mM DTT for 60 minutes at 37° C. PAD2 treated myocytes were then exposed to a 1 : 10 dilution of activated PAD2 in isolation buffer for 30 minutes at room temperature. Myocytes were then glued with silicone to the tips of 150 μπι diameter minutia pins attached to a force transducer and motor arm (Aurora Scientific Inc., Aurora, ON, Canada). Sarcomere length was monitored by video camera (Imperx, Boca Raton, FL) and calculated by the High-speed Video Sarcomere Length Program (Aurora Scientific Inc.). Myocyte sarcomere length was set at 2.1 μπι. A complete activation of the myocyte occurred at the beginning and end of the experiment, and the myocyte discarded if there was >10% rundown, as described [Kirk JA et al. Cardiac resynchronization sensitizes the sarcomere to calcium by reactivating GSK-3beta. J Clin Invest. 2014, 1 : 129-38]. ATPase activity

[0095] The HMM- ATPase activity was analyzed at three separate experiments described previously [White HD. Special instrumentation and techniques for kinetic studies of contractile systems. Methods Enzymol 1982, 85:698-708; Tauhata SB, et al. High affinity binding of brain myosin- Va to F-actin induced by calcium in the presence of ATP. J Biol Chem 2001, 276:39812-39818]. First experiment was carried at constant HMM and F-actin concentration with citrullinated or non-citrullinated HMM and/or F-actin. Second, HMM- ATPase activity was determined at increasing TM concentrations (citrullinated or non- citrullinated). Third experiment HMM-ATPase activity was determined at constant HMM, F- actin and TM concentration with citrullinated or non-citrullinated Tnl. Each experiment was done in triplicate and three separate times.

Actin Binding Experiments

[0096] Various concentrations of citrullinated and non-citrullinated HMM, TM and/or Tnl were added to F-actin and centrifuged to determine extent of binding. Pellet and supernatant were analyzed using 10% SDS PAGE, and amount of each protein quantified by densitometry as previously described [Skorzewski R et al. Effect of actin C-terminal modification on tropomyosin isoforms binding and thin filament regulation. Biochim Biophys Acta 2009, 2: 237-243; Nascimento AA et al. Enzymatic characterization and functional domain mapping of brain myosin-V. J Biol Chem 1996, 271 : 17561-17569]. Each assay was carried out in triplicate.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

[0097] Total mRNA from mouse neonatal cardiac myocytes was extracted using TRI-reagent (Sigma) according to the manufacturer's protocols. Complementary DNA (cDNA) was generated using the Superscript III First-Standard Synthesis System (Invitrogen) according to the instructions of the manufacturer. RT-PCR was performed using primers specific for the PAD1, PAD2, PAD3, PAD4 and β-actin. The PCR products were separated by electrophoresis on a 1.8% agarose gel and visualized under UV light. Each assay was done in duplicate. ID-PAGE and Western Blot

[0098] Proteins lOug per well were separated by ID-PAGE using 12-well 4-12% NuPAGE Bis-Tris gels and blotted onto PVDF membranes according to manufacturer's instruction. After staining the blotted membrane with Direct Blue followed by scanning images, membranes were incubated with modification reagent [1 vol of 1% diacetyl monoxime/0.5% antipyrine/1 N acetic acid, and 2 vol of a mixture of 85% H ₃ P0 ₄ /98% H ₂ S0 ₄ /H ₂ 0 (20/25/55) containing 0.025%) FeCl ₃ ] overnight at 37°C, protected from light. The membrane was blocked, probed overnight at 4°C with an anti-modified citrulline antibody at 1 :2000 (Millipore, Billerica, MA) and followed by washes incubated with the secondary antibody for 1 hour (goat polyclonal antirabbit IgG, 1 : 10000 dilution). Bound antibodies were visualized by chemiluminescent substrate as described by the manufacturer (Amersham Biosciences, Piscataway, NJ). When developed, films were scanned with Epson Expression 10000 XL scanner (Epson, US) and imported into GraphPad Prism tools. A student's t-test was used to determine statistical difference between the groups. A p-value < 0.05 was considered statistically significant.

Sample deimination, Cy labeling, 2-DE separation

[0099] Heart samples, weighing approximately 150 mg, were subfractionated as described earlier. For deimination of each fraction was treated with 1.5mg bacterial PAD2 in 200mM Tris-HCl pH 7.5/10mM dithiothreitol (DTT)/20mMCaCl ₂ in a total volume of lOOuL. The same amount of each sample was treated at the same condition but without enzyme. After incubating the samples at 50°C for 2 hours samples were mixed with DIGE labeling buffer (7 M urea/2 M thiourea/4% CHAPS/30 mM Tris pH 8.5) at a final concentration of 70ug/110uL. Cy dyes were added (3200 pmol/400 mg aliquot) and aliquots were incubated at RT in the dark for 30 min. Reactions were stopped by adding 1 mL lOmM lysine per 400 pmol dye. Pooled standard was labeled with Cy2, samples no PAD2 treated were labeled with Cy3, and samples treated with PAD2 were labeled with Cy5. A 150ug aliquot of pooled standard, sample +PAD2 and sample - PAD2, labeled as stated above, were mixed together and diluted in IEF buffer (7 M urea/2 M thiourea/4%CHAPS/l% DTT/1% 3-10 ampholytes) to final volume 350ul. Prepared samples were applied to 18 cm immobilized linear pH gradients (4-7) strips (IPG, BioRad), with active rehydration for 12 h at 20°C. For isoelectrofocusing (IEF), the BioRad Protean IEF cell was used with the following conditions at 20°C: Step 1 : lhr with end voltage at 200 V; Step 2: lh ramping with end voltage at 500 V; Step 3 : 1 hr with end voltage 500V; Step 4: 1 hr ramping with end voltage at 1000 V; Step 5: 2 hr with end voltage 1000V; Step 6: 2 hrs. ramping with end voltage at 1000 V; Step 7: 50000V/h (approximately 6hrs). After IEF, the strips were equilibrated according to the manufacturer's instructions. Second dimension of 2-DE was then carried out with 10% gels and electrophoresed at constant voltage (80 V) for 30-60 min, followed by constant power (200V, 200 mA) for 6 hours. The DIGE gels were scanned using a Typhoon 9410 (GE Healthcare) at different emission wavelengths, which allows the differentially expressed proteins to be viewed as changed in color. Preparative gels were stained with silver staining and scanned with the Typhoon 9410.

Image Analysis

[0100] Small gels were scanned using an Epson Expression 10000 XL (Epson America), and large gels were scanned using an Amersham Typhoon 9410 (GE Healthcare). Differentially expressed proteins (treated vs. no treated with PAD2) were identified using Ludesi REDFIN analysis (Ludesi, http://www.ludesi.com) for spot detection, matching, and analysis. Ludesi REDFIN analysis was performed in two steps: differential in-gel analysis (DIA) followed by biological variation analysis (BVA). DIA quantifies protein volume from fluorescence intensity for Cy2, Cy3 and Cy5 and calculates the ratios Cy3/Cy2 (no PAD2 treated to internal standard) and Cy5/Cy2 (PAD2 treated to internal standard). Values are then normalized based on the assumption that the amount of protein per image is the same. 2-DE gel images were analyzed in REDFINA (http://www.ludesi.com) using student's t-test to determine statistical difference between the groups. A p-value < 0.05 was considered statistically significant. Spots of interest were then excised from gel, destined and in-gel digested with trypsin. Digested samples were used for LC-MS/MS analysis as described below.

In-gel protein digestion and peptide identification

[0101] Spots of interest were picked out from the gels with a shortened pipette tip, placed in small eppendorf tubes and distained by using a 30 mM potassium ferricyanide and 100 mM sodium thiosulfate. After a further washing with bi-distilled water, gel plugs were dried and rehydrated directly in reducing and alkylate reagent follow by digestion of 0.1 μg/μl trypsin in 50 mM H ₄ HCO ₃ pH 8.8 solution (Trypsin ultra grade sequencing grade, Promega, WI, USA). Digestion was performed lhr on ice; next the extra solution was removed and gel plugs were cover with lOOul 50 mM H ₄ HCO ₃ and incubate overnight at 37°C. Peptides were desalted by ZipTip and analyzed by nanoflow LC-MS/MS by using an LTQ ion trap MS (Thermo Fisher Scientific) interfaced with an Agilent 1200 nanoLC system.

Mass Spectrometry

[0102] Each human heart was fractionated into myofilament- and cytosolic-enriched fractions using EST Sequence protocol [Kane LA, et al. Subfractionation of heart tissue: the "in sequence" myofilament protein extraction of myocardial tissue. Methods Mol Biol 200, 357:87-90]. Protein extracts were denatured, reduced and alkylated prior to digestion with LysC (1 :20) using a filter-aided sample preparation (FASP) protocol (https://www.biochem.mpg.de/226356/FASP). Exogenous internal retention time standards (IRT, company and reference [Escher C, et al. Using iRT, a normalized retention time for more targeted measurement of peptides. Proteomics 2012, 12: 1111-1121]) were added to each peptide fraction. To maximize the citrullinated proteome depth in vitro citrulllination, using PADs cocktail, of cardiac tissue lysates was performed followed by the proteins digestion with LysC. Spectral ion libraries were constructed by data dependent acquisition on an AB Sciex 6600 TripleTOF and the raw data was searched with ProteinPilot™ Software 5.0 to create a spectral ion library. The two fractions from each individual were sequentially analyzed using data independent acquisition (SWATH-MS) on the 6600 Triple TOF. The peptide data were kept for further analysis if they contained at least one high confidence citrulline-peptide/spectrum assignment (>95% confidence) and the confidence level of the protein to which it mapped was >95%. Peptide and ProteinProphet thresholds, the global false discovery rates for this study, obtained from a search of the reversed human database, were estimated to be on the order of <0.1% at the peptide and protein levels respectively. Importantly, transitions of each citrullinated peptide were selected and verified to provide accurate sensitivity and specificity for these targeted proteomics experiments. SWATH-MS fragment ion maps were created (from the endogenous samples) between 400-1250 m/z using 200 variable SWATH windows with a dwell time of 20 ms for each window. Source conditions were as follows for DDA and SWATH experiments: Spray voltage was set to 2.4 kV, source gas was set to 5, curtain gas was set to 30, interface heater temperature was set to 80, and declustering potential was set to 100. Rolling collision energy was used for MS2 experiments in the DDA runs. For both DDA and SWATH runs peptides were separated using an Eksigent ekspert™ 415 nanoLC equipped with ekspert™ cHiPLC and ekspert™ nanoLC 400 autosampler. Samples were loaded onto a trap column (nano cHiPLC Trap 200 μπι x 0.5 mm ChromXP C18-CL 3 μπι 120 A) for 10 minutes at a flow rate of 2 μΕ/πήη in 100% solvent A (0.1% formic acid in water) after which point they were separated using a nano cHiPLC 75 μιη x 15 cm ChromXP C18-CL 3 μιη 120 A column using a flow rate of 300 nL/min and a linear gradient of 5-35% solvent B (0.1% formic acid in acetonitrile) for 120 min, 35-85% B for 2 minutes, holding at 85% for 5 minutes, then re-equilibration at 5% B for 17 minutes.

Construction of a verified citrullinated spectral ion library

[0103] A verified citrullinated peptide ion library was constructed based on the retention time (RT) difference between pairs of peptides that had un-modified (non-citrullinated) and citrullinated forms present in the ion library and between peptides that had N/Q deaminated and citrullinated forms. Based on our data we observed that the difference in RT between the unmodified and citrullinated form of the same peptide sequence (in the absence of any other modifications) was on average 10 minutes, with the citrullinated peptide having the longer retention time, whereas N/Q deamidation resulted in a RT shift on average of about 2.5 minutes. Therefore, for citrullinated peptide sequences that had paired unmodified forms or paired N/Q deamidation forms and the RT difference was greater than 5 minutes we did not alter the selected transition ions in the ion library. For citrullinated peptides where the RT was less than 5 minutes between the pairs or if there was no unmodified form to use as a reference we manually selected transition ions that would unambiguously distinguish the citrullinated peptide from the unmodified or N/Q deaminated form and any transitions that did not unambiguously distinguish the citrullination site were removed from the ion library. For this subset of peptides five unique transitions were required for a citrullinated peptide to be included in the library. In addition to the verified citrullinated peptides for each protein, a maximum of 10 unmodified peptides were incorporated into the library for total protein quantification.

SWATH data analysis

[0104] The verified ion library was imported into Peak View v2.1 and SWATH files were loaded into the SWATH microapp version 2.0. Extraction settings were as follows: 30 peptides per protein, 5 transitions per peptide, 95% peptide confidence cutoff, an FDR threshold of 1 %, an XIC extraction window of 5 min and tolerance of 0.05 Da. Co-sedimentation assay

[0105] Binding assays were performed by TM cosedimentation (0-2uM) with 5uM F-actin essentially as described earlier [Skorzewski R, et al. Effect of actin C-terminal modification on tropomyosin isoforms binding and thin filament regulation. Biochim Biophys Acta, 2009, 2:237-243], with the following modifications. Binding of TM alone was assayed in 40mM Tris HC1, pH 7.6, 1 mM DTT, 5 mM MgCl ₂ , and 100 mM NaCl. After mixing of all assay components, samples were incubated at room temperature for ~0.5 h to ensure attainment of the steady-state. Protein mixtures were ultracentrifuged for 25 min at 60,000 rpm in Beckman rotor TLA 100.2. The composition of the proteins in pellets and the amount of free TM left in the supernatant were examined on 4-12% SDS-PAGE. The gels stained with Coomassie Blue were scanned and quantified. Intensities of SDS-gel bands of pelleted F- actin were similar. TM bound to actin was calculated as TM/actin band intensity ratio normalized to the maximum ratio reached at saturation. The concentration of unbound TM was calculated from band intensities of TM left in the supernatant.

Myosin HMM-induced binding of TM to F-actin

[0106] F-actin (5 μΜ) and TM (1 μΜ, citrullinated or control) in 30 mM NaCl, 0.5 mM MgC12, 1 mM DTT, 5 mM imidazole, pH 7.0, were mixed with HMM (0-3.5 μΜ). The mixture was incubated at room temperature for 30 min and then centrifuged in a TLA-100.2 rotor for 25 min, at 60,000 rpm at 15°C. The composition of proteins sedimented in pellets was analyzed by SDS-PAGE and densitometry [Sliwinska M, et al. Role of actin C-terminus in regulation of striated muscle thin filament. Biophys J. 2008, 4:1341-].

[0107] FDVIM-ATPase assays. The assays were performed as described by White [White FID. Special instrumentation and techniques for kinetic studies of contractile systems. Methods Enzymol 1982, 85:698-708] with several modifications. All assays were carried out at 22° C in a buffer containing 30 mM NaCl, 5mM MgC12, and lOmM Hepes, pH 7.5 with F-actin 0.2 mg/ml and HMM 0.02 mg/ml final concentrations. When present, non-citrullinated or citrullinated TM was added to F-actin to a final molar concentrations between 0-2 μΜ TM. HMM-Actin-TM-Tnl assay was carried as above with citrullinated or not citrullinated Tnl at F-actin:TM:TnI molar ration 7: 1 : 1 respectively. To assess the sensitivity of the regulated acto-Sl system, all assays were done in parallel both in the presence (0.5 mM CaC12,) and the absence (2 mM EGTA) of calcium. All reactions were initiated by addition of 4 mM ATP. The amount of liberated phosphate was determined calorimetrically at 650 nm. All assays were carried out within the linear range and each assay was done in triplicate.

Example 2

Identification of myocardial citrullinated proteins.

[0108] To identify citrullinated targets in the heart we assessed the citrullinome in three groups, ISHD, IDCM and non-failing donor hearts (n=10 per group) using SWATH-MS [34]. SWATH-MS allowed for the quantification. 53 citrullinated sites were altered with HF compared to the non-failing controls (p<0.05) and are listed in Table 1 A.

[0109] Table 1A: List of citrullinated protein (p=0.05) with citrullinated peptides sequence. "Dea" is Citrullination or deamination. "CAM" is carbamidomethylation. Citrullinated proteins were grouped by cellular component. UP identifier = Universal Protein (UP)Resource protein ID; p kw = p value for Kruskal-Wallis test (statistic)

UniProtKB

Protein Peptide p kw

Accessions

Troponin I, cardiac

NIDALSGMEGR[Dea]K (SEQ ID NO: 18) TNNI3 0.02 muscle

Troponin I, cardiac

ESLDLR[Dea]AHLK (SEQ ID NO: 19) TNNI3 0.02 muscle

Troponin T, cardiac

PR[Dea] SFMPNL VPPK (SEQ ID NO: 20) E7EPW4 0.01 muscle

Tropomyosin ** ETR[Dea]AEFAERSVTKLEK (SEQ ID NO: 21) TPM1 0.002

FADLSEAAN[Dea]RNNDALR[Dea]QAK (SEQ

Vimentin VIME 0.03

ID NO: 22)

Gly ceraldehy de-3 - LWR[Dea]D[Dhy] GRGALQN[Oxi]IIPASTGAAK

G3P 0.01 phosphate dehydrogenase (SEQ ID NO: 23)

AEETQR[Dea] S VNDLTSQR[Dea] AK (SEQ ID

Myosin-7 MYH7 0.05

NO: 24)

SYLRpea]LRPDRVAMQDATAQ[Dea]M[Oxi]A

Aconitate hydratase, ACON 0.01

MLQFISSGLSK (SEQ ID NO: 25)

ANSVR[Dea]NAVTQEFGPVPDTAR[Dea]YYK

Aconitate hydratase, ACON 0.03

(SEQ ID NO: 26)

IVYGHLDDPASQEIER[Dea]GK (SEQ ID NO:

Aconitate hydratase, ACON 0.03

27)

Adenylate kinase 4, LLR[Dea]AVILGPPGSGK (SEQ ID NO: 28) KAD4 0.00

Alcohol dehydrogenase

VAGASR[Dea]IIGVDINK (SEQ ID NO: 29) ADHX 0.05 class-3

ATP synthase subunit R[Dea]TGAIVDVPVGEELLGR[Dea]WDALGN

ATPA 0.00 alpha, AIDGK (SEQ ID NO: 30)

ATP synthase subunit AIEEQVAVIYAGVR[Dea] GYLDK (SEQ ID NO:

ATPA 0.01 alpha, 31)

ATP synthase subunit GIPJ>AINVGLSVSR[Dea]VGSAAQ[Dea]TRAMK

ATPA 0.01 alpha, (SEQ ID NO: 32)

ATP synthase subunit QGQYSPMAIEEQVAVIYAGVR[Dea] GYLDK

ATPA 0.03 alpha, (SEQ ID NO: 33)

ATP synthase-coupling SGGPVDASSEYQQELER[Dea]ELFK (SEQ ID

ATP5J 0.00 factor 6, NO: 34)

Chloride intracellular

YR[Dea]NFDIPK (SEQ ID NO: 35) CLIC4 0.04 channel protein 4

Cytochrome c (Fragment) EER[Dea]ADLIAYLK (SEQ ID NO: 36) C9JFR7 0.01

Delta- 1 -pyrroline-5 - carboxylate VLR[Dea]NAAGNFYINDK (SEQ ID NO: 37) AL4A1 0.00 dehydrogenase,

Delta- 1 -pyrroline-5 - AADMLSGPR[Dea]R[Dea]AEILAK (SEQ ID NO:

carboxylate AL4A1 0.01

38)

dehydrogenase,

Delta- 1 -pyrroline-5 - VANEPVLAFTQGSPER[Dea]DALQK (SEQ ID

carboxylate AL4A1 0.03

NO: 39)

dehydrogenase,

Enoyl-CoA hydratase, EMVLTGDR[Dea]ISAQDAK (SEQ ID NO: 40) ECHM 0.01

VLR[Dea]GVEVTVGHEQEEGGK (SEQ ID NO:

ESI protein homolog, ES I 0.00

41)

Heat shock protein HSP

HLEINPDHSIIETLR[Dea]QK (SEQ ID NO: 42) HS90A 0.04 90-alpha

NAD(P)

AATITPFR[Dea]K (SEQ ID NO: 43) NNTM 0.05 transhydrogenase, UniProtKB

Protein Peptide p kw

Accessions

Phosphate carrier protein, VYFR[Dea]LPRPPPPEMPESLK (SEQ ID NO: 44) MPCP 0.00

Pyruvate dehydrogenase C[CAM]DLHR[Dea]LEEGPPVTTVLTR[Dea]ED

El component subunit ODPA 0.01

GLK (SEQ ID NO: 45)

alpha, somatic form,

Succinate dehydrogenase

[ubiquinone] iron-sulfur FAIYR[Dea]WDPDK (SEQ ID NO: 46) SDHB 0.05 subunit,

Succinyl-CoA ligase TR[Dea]LIGPNC [C AMJPGVINPGEC [C AM]K

[ADP/GDP-forming] SUCA 0.02

(SEQ ID NO: 47)

subunit alpha,

Succinyl-CoA:3-ketoacid ADR[Dea]AGNVIFR[Dea]K (SEQ ID NO: 48) SCOT1 0.04 coenzyme A transferase 1,

Voltage-dependent anion- selective channel protein SR[Dea]VTQSNFAVGYK (SEQ ID NO: 49) VDAC1 0.02 1

60 kDa heat shock FDR[Dea] GYISPYFINTSK (SEQ ID NO: 50) CH60 0.04 protein,

Trifunctional enzyme PNIR[Dea]NVVWDGVR[Dea]TPFLLSGTSYK

ECHB 0.02 subunit beta, (SEQ ID NO: 51)

Aconitate hydratase, TGR[Dea]EDIANLADEFK (SEQ ID NO: 52) ACON 0.00

Aconitate hydratase, R[Dea]LQLLEPFDK (SEQ ID NO: 53) ACON 0.01

Dual specificity protein LGITHVLNAAEGR[Dea] SFMHVNTNANFYK

DUS3 0.01 phosphatase 3 (SEQ ID NO: 54)

Neuroblast

differentiation-associated FGVSTGR[Dea]EGQTPK (SEQ ID NO: 55) AHNK 0.01 protein AHNAK

Collagen alpha-3( VI) D WFLLDGSEGVR[Dea] SGFPLLK (SEQ ID

E7ENL6 0.03 chain NO: 56)

[0110] Table IB shows all citrullinated peptides found in this study. These were proteins with diverse cellular functions, including the regulators of transcription and chromatin structure, cytoskeletal and contraction, cellular signaling processes and metabolism (Figure 1).

[0111] Table IB: all citrullinated peptides found in this study. "Dea" is Citrullination or deamination. "CAM" is carbamidomethylation.

Protein Peptide UniProtKB p kw

Accessions

Adenylate kinase isoenzyme LLR[Dea]SEVSSGSAR[Dea]GK (SEQ ID NO: 60) Q5T9B7 0.68 1

Adenylate kinase isoenzyme YGYTHLSTGDLLR[Dea] SEVSSGSARGK (SEQ ID Q5T9B7 0.74 1 NO: 61)

Alcohol dehydrogenase IB AAGAAR[Dea] II AVDINK (SEQ ID NO: 2) P00325 0.04

Aldose reductase PEDPSLLEDPR[Dea]IK (SEQ ID NO: 62) P15121 0.18

Alpha-crystallin B chain GLSEMR[Dea]LEK (SEQ ID NO: 10) P02511 0.01

Alpha-crystallin B chain QVSGPER[Dea]TIPITREEK (SEQ ID NO: 63) P02511 0.23

Alpha-crystallin B chain YR[Dea]IPADVDPLTITSSLSSDGVLTVNGPR[Dea P02511 0.41

]K (SEQ ID NO: 64)

Alpha-crystallin B chain SPFYLR[Dea]PPSFLR[Dea]APSWFDTGLSEMRLE P02511 0.51

K (SEQ ID NO: 65)

Alpha-crystallin B chain SPFYLRPPSFLR[Dea]APSWFDTGLSEMRLEK P02511 0.55

(SEQ ID NO: 66)

Alpha-crystallin B chain QVSGPER[Dea]TIPITR[Dea]EEK (SEQ ID NO: 67) P02511 0.97

Aspartate aminotransferase, HIYLLPSGR[Dea]INVSGLTTK (SEQ ID NO: 68) P17174 0.84

ATP-dependent 6- LLAHVR[Dea]PPVSK (SEQ ID NO: 69) P08237 0.53 phosphofructokinase

ATP-dependent 6- NVLGHMQQGGSPTPFDR[Dea]NFATK (SEQ ID P08237 0.80 phosphofructokinase NO: 70)

ATP-dependent 6- [ 1 Ac] -MD ADD SR[Dea] APK (SEQ ID NO: 71) Q01813 0.55 phosphofructokinase

Beta-enolase SPDDPAR[Dea]HITGEK (SEQ ID NO: 3) P13929 0.00

Carbonic anhydrase 3 DIR[Dea]HDPSLQPWSVSYDGGSAK (SEQ ID NO: P07451 0.01

4)

Creatine kinase M-type ELFDPIISDR[Dea]HGGYK (SEQ ID NO: 72) P06732 0.20

Creatine kinase M-type GGDDLDPNYVLSSR[Dea]VR[Dea]TGR[Dea]SIK P06732 0.36

(SEQ ID NO: 73)

Creatine kinase M-type PIISDR[Dea]HGGYKPTDK (SEQ ID NO: 74) P06732 0.47

Creatine kinase M-type ELFDPIISDR[Dea]HGGYKPTDK (SEQ ID NO: 75) P06732 0.51

Creatine kinase M-type GGDDLDPNYVLSSRVR[Dea]TGRSIK (SEQ ID P06732 0.66

NO: 76)

Creatine kinase M-type FEEILTR[Dea]LRLQK (SEQ ID NO: 77) P06732 0.82

Fatty acid-binding protein LILTLTHGTAVC[C AM] TR[Dea] TYEK (SEQ ID P05413 0.04

NO: 5)

Glyceraldehyde-3 -phosphate LWR[Dea]D[Dhy] GRGALQN[Oxi]IIPASTGAAK P04406 0.01 dehydrogenase (SEQ ID NO: 23)

Glycogen phosphorylase VAIQLNDTHP AL SIPELMR[Dea] IL VD VEK (SEQ P11216 0.39

ID NO: 78)

Glycogen phosphorylase QIS VR[Dea] GLAGLGD VAEVRK (SEQ ID NO: 79) P11216 0.63

Hemoglobin subunit alpha LR[Dea]VDPVN[Dea]FK (SEQ ID NO: 80) P69905 0.91

Hemoglobin subunit alpha VGAHAGEYGAEALER[Dea]MFLSFPT (SEQ ID P69905 0.93

NO: 81)

Hemoglobin subunit alpha LER[Dea]MFLSFPTTK (SEQ ID NO: 82) P69905 0.93

Hemoglobin subunit alpha R[Dea]VDPVNFK (SEQ ID NO: 83) P69905 0.93

Hemoglobin subunit beta VNVDEVGGEALGRLLWYPWTQ[Dea]R[Dea]FF P68871 0.40 Protein Peptide UniProtKB p kw

Accessions

ESFGDLSTPDAVM[Oxi]GNPK (SEQ ID NO: 84)

Hemoglobin subunit beta VNVDEVGGEALGR[Dea]LLV (SEQ ID NO: 85) P68871 0.46

Hemoglobin subunit beta VNVDEVGGEALGR[Dea]LLWYPWTQ[Dea]RFF P68871 0.67

ESFGDLSTPDAVMGNPK (SEQ ID NO: 86)

Hemoglobin subunit beta R[Dea]FFESFGDLSTPDAVMGNPK (SEQ ID NO: P68871 0.82

87)

Hemoglobin subunit beta VNVDEVGGEALGR[Dea]L (SEQ ID NO: 88) P68871 0.90

Peptidyl-prolyl cis-trans TAENFR[Dea] AL STGEK (SEQ ID NO: 6) P62937 0.01 isomerase A

Phosphatidylethanolamine- VLTDPDAPSR[Dea]KDPK (SEQ ID NO: 89) P30086 0.12 binding protein 1

Phosphatidylethanolamine- C[CAM]DEPILSN[Dea]RSGDHR[Dea]GK (SEQ ID P30086 0.55 binding protein 1 NO: 90)

Phosphoglycerate kinase 1 SWLMSHLGR[Dea]PDGVPMPDK (SEQ ID NO: P00558 0.26

91)

Phosphoglycerate kinase 1 R[Dea]WMR[Dea]VDFNVPMK (SEQ ID NO: 92) P00558 0.27

Serum albumin YLYEIARR[Dea]H[AAS]P[Oxi]YFYAPELLFFAK P02768 0.06

(SEQ ID NO: 93)

Serum albumin VPQVSTPTLVEVSR[Dea]N (SEQ ID NO: 94) P02768 0.31

Serum albumin WAVAR[Dea]LSQR[Dea]FPK (SEQ ID NO: 95) P02768 0.40

Serum albumin FGER[Dea]AFK (SEQ ID NO: 96) P02768 0.46

Serum albumin TESLVNR[Dea]RPC[CAM] (SEQ ID NO: 97) P02768 0.49

Serum albumin AWAVAR[Dea]LSQR[Dea]FPK (SEQ ID NO: 98) P02768 0.64

Serum albumin DDNPNLPP VR[Dea]PEVD VMC [CAM] TAFHDN P02768 0.66

EETFLK (SEQ ID NO: 99)

Serum albumin TPVSDR[Dea]VTK (SEQ ID NO: 100) P02768 0.69

Serum albumin ALLVR[Dea]YTK (SEQ ID NO: 101) P02768 0.70

Serum albumin SEVAHR[Dea]FK (SEQ ID NO: 102) P02768 0.72

Serum albumin LVEVSR[Dea]NLGK (SEQ ID NO: 103) P02768 0.74

Serum albumin R[Dea]MPC[CAM]AEDYLSWLNQLC[CAM]VLH P02768 0.82

EK (SEQ ID NO: 104)

Serum albumin DDNPNLPR[Dea]LVR[Dea]PEVDVMC[CAM]TAF P02768 0.84

HDNEETFLK (SEQ ID NO: 105)

Serum albumin [CRM] -YLYEIAR[Dea]RHPYFYAPELLFFAK P02768 0.86

(SEQ ID NO: 106)

Serum albumin C[CAM]C[CAM]TESLVNR[Dea]RPC[CAM] (SEQ P02768 0.93

ID NO: 107)

Serum deprivation-response FQHPGSDMR[Dea]QEK (SEQ ID NO: 7) 095810 0.01 protein

Serum deprivation-response VSPLTFGR[Dea]K (SEQ ID NO: 8) 095810 0.01 protein

Transgelin R[Dea]EFTESQLQEGK (SEQ ID NO: 108) Q01995 0.22

Translationally-controlled MIIYR[Dea]DLISHDEMFSDIYK (SEQ ID NO: 109) Q5W0H4 0.14 tumor protein

Tumor protein D54 TSAALSTVGSAISR[Dea]K (SEQ ID NO: 110) 043399 0.08

Malate dehydrogenase FVEGLPINDFSR[Dea]EK (SEQ ID NO: 111) P40925 0.17 Protein Peptide UniProtKB p kw

Accessions

Actin, alpha skeletal muscle C[CAM]DIDIR[Dea]K (SEQ ID NO: 11) Q5T8M8 0.11

Actin, alpha skeletal muscle QEYDEAGPSIVHR[Dea]K (SEQ ID NO: 12) Q5T8M8 0.16

Desmin EEAENNLAAFR[Dea]ADVDAATLAR (SEQ ID P17661 0.06

NO: 112)

Desmin SR[Dea]LGTTR[Dea]TPSSYGAGELLDFSLADAV P17661 0.08

NQEFLTTRTNEK (SEQ ID NO: 113)

Desmin NFRETSPEQR[Dea]GSEVHTK (SEQ ID NO: 114) P17661 0.23

Desmin MALDVEIATYR[Dea]K (SEQ ID NO: 115) P17661 0.23

Desmin SR[Dea]LGTTRTPSSYGAGELLDFSLADAVNQEF P17661 0.31

LTTRTNEK (SEQ ID NO: 116)

Desmin VDVER[Dea]DNLLDDLQRLK (SEQ ID NO: 117) P17661 0.39

Desmin NFR[Dea]ETSPEQR[Dea]GSEVHTK (SEQ ID NO: P17661 0.43

118)

Desmin TIETR[Dea]DGEWSEATQQQHEVL (SEQ ID NO: P17661 0.55

119)

Desmin S SP VFPR[Dea] AGFGSK (SEQ ID NO: 120) P17661 0.88

Destrin [ 1 Ac] - ASGVQ VADEVC [C AM]R[Dea] IFYDMK P60981 0.78

(SEQ ID NO: 121)

Filamin-C SSSSR[Dea]GSSYSSIPK (SEQ ID NO: 13) Q14315 0.03

Four and a half LIM domains KPITTGGVTYR[Dea]EQPWHK (SEQ ID NO: 122) Q14192 0.08 protein 2

Fructose-bisphosphate EL SDI AHR[Dea] I VAPGK (SEQ ID NO: 123) P04075 0.55 aldolase A

Glyceraldehyde-3 -phosphate LVIN[Dea]GNPITIFQER[Dea]DPSK (SEQ ID NO: P04406 0.66 dehydrogenase 124)

Glyceraldehyde-3 -phosphate R[Dea]VIISAPSADAPMFVMGVNHEK (SEQ ID P04406 0.68 dehydrogenase NO: 125)

Glyceraldehyde-3 -phosphate AFRVPTAN[Dea]VSWDLTC[CAM]R[Dea]LEK P04406 0.88 dehydrogenase (SEQ ID NO: 126)

Glyceraldehyde-3 -phosphate VSWDLTC[CAM]R[Dea]LEKPAK (SEQ ID NO: P04406 0.93 dehydrogenase 127)

Glyceraldehyde-3 -phosphate LWR[Dea]DGR[Dea] GALQNIIPASTGAAK (SEQ P04406 0.99 dehydrogenase ID NO: 128)

LIM domain-binding protein TSPEGAR[Dea]DLLGPK (SEQ ID NO: 14) 075112 0.00

3

Moesin DR[Dea]SEEER[Dea]TTEAEK (SEQ ID NO: 129) P26038 0.28

Myomesin-1 HVSGITDTEEER[Dea]IK (SEQ ID NO: 130) P52179 0.07

Myomesin-1 SPR[Dea]FALFDLAEGK (SEQ ID NO: 131) P52179 0.57

Myosin light chain 3 LGQNPTQAEVLR[Dea]VLGKPRQEELNTK (SEQ P08590 0.09

ID NO: 132)

Myosin light chain 3 LGKPR[Dea]QEELNTK (SEQ ID NO: 133) P08590 0.16

Myosin light chain 3 [CRM]-EAFMLFDR[Dea]TPK (SEQ ID NO: 134) P08590 0.39

Myosin light chain 3 EAFMLFDR[Dea]TPK (SEQ ID NO: 135) P08590 0.41

Myosin light chain 3 VLGKPR[Dea] QEELNTK (SEQ ID NO: 136) P08590 0.42

Myosin light chain 3 ALGQNPTQAEVLR[Dea]VLGK (SEQ ID NO: 137) P08590 0.59

Myosin light chain 3 LGQNPTQAEVLR[Dea]VLGKPRQ[Dea]EELNTK P08590 0.70 Protein Peptide UniProtKB p kw

Accessions

(SEQ ID NO: 138)

Myosin light chain 3 LGQNPTQAEVLR[Dea]VLGK (SEQ ID NO: 139) P08590 0.71

Myosin light chain 3 EAFM[Oxi]LFDR[Dea]TPK (SEQ ID NO: 140) P08590 0.86

Myosin light chain 4 MLDFETFLPILQHISR[Dea]NK (SEQ ID NO: 141) P12829 0.28

Myosin light chain 4 EQGTYEDFVEGLR[Dea]VFDK (SEQ ID NO: 142) P12829 0.74

Myosin regulatory light chain NDLRDTFAALGR[Dea]VNVK (SEQ ID NO: 143) P10916 0.71 2

Myosin regulatory light chain EAFTIMDQNR[Dea]DGFIDK (SEQ ID NO: 144) P10916 0.77 2

Myosin-7 AEETQR[Dea]SVNDLTSQR[Dea]AK (SEQ ID NO: P12883 0.05

24)

Myosin-7 [ 1 Ac] -GD SEMAVFGAAAPYLR[Dea]K (SEQ ID P12883 0.18

NO: 145)

Myosin-7 VR[Dea]ELENELEAEQK (SEQ ID NO: 146) P12883 0.27

Myosin-7 R[Dea]YR[Dea]ILNPAAIPEGQFIDSRK (SEQ ID P12883 0.28

NO: 147)

Myosin-7 RNHLR[Dea]WDSLQT (SEQ ID NO: 148) P12883 0.31

Myosin-7 R[Dea]VR[Dea]ELENELEAEQK (SEQ ID NO: 149) P12883 0.40

Myosin-7 AVFGAAAPYLR[Dea]K (SEQ ID NO: 150) P12883 0.42

Myosin-7 LEAR[Dea]VR[Dea]ELENELEAEQK (SEQ ID NO: P12883 0.43

151)

Myosin-7 YR[Dea] ILNP AAIPEGQFID SRK (SEQ ID NO: 152) P12883 0.51

Myosin-7 RYR[Dea] ILNP AAIPEGQFID SRK (SEQ ID NO: P12883 0.52

153)

Myosin-7 DFELNALNAR[Dea]IEDEQALGSQLQK (SEQ ID P12883 0.58

NO: 154)

Myosin-7 AEETQRSVNDLTSQR[Dea]AK (SEQ ID NO: 155) P12883 0.60

Myosin-7 RSVNDLTSQR[Dea]AK (SEQ ID NO: 156) P12883 0.74

Myosin-7 R[Dea]VRELENELEAEQK (SEQ ID NO: 157) P12883 0.76

Myosin-7 YRILNP AAIPEGQFID SR[Dea]K (SEQ ID NO: 158) P12883 0.81

Myosin-7 RVIQYFAVIAAIGDR[Dea] SK (SEQ ID NO: 159) P12883 0.86

Myosin-7 RYR[Dea]ILNPAAIPEGQFIDSR[Dea]K (SEQ ID P12883 0.96

NO: 160)

Myosin-binding protein C EPVFIPR[Dea]PGITYEPPNYK (SEQ ID NO: 15) A8MXZ9 0.03

Myosin-binding protein C VRWQR[Dea]GGSDISASNK (SEQ ID NO: 161) A8MXZ9 0.09

Myosin-binding protein C PR[Dea]PQVTWTK (SEQ ID NO: 162) A8MXZ9 0.21

Myozenin-2 R[Dea]VATPFGGFEK (SEQ ID NO: 16) Q9NPC6 0.01

Myozenin-2 AELPDYR[Dea] SFNR[Dea] VATPFGGFEK (SEQ Q9NPC6 0.02

ID NO: 17)

Phosphoglucomutase- 1 SGEHDFGAAFDGDGDR[Dea]NMILGK (SEQ ID P36871 0.86

NO: 163)

Phosphoglucomutase- 1 DLEALMFDR[Dea] SFVGK (SEQ ID NO: 164) P36871 0.87

Plastin-2 VNDDIIVNWVNETLR[Dea]EAKK (SEQ ID NO: P13796 0.45

165) Protein Peptide UniProtKB p kw

Accessions

Profilin-1 C[CAM]SVIR[Dea]DS[Dhy]LLQDGEFSMDLRTK P07737 0.18

(SEQ ID NO: 166)

Titin C[CAM]DVS[Dhy]R[Dea]GDWVTALASVTK (SEQ Q8WZ42 0.17

ID NO: 167)

Tropomyosin* ETR[Dea]AEFAERSVTKLEK (SEQ ID NO: 21) B7Z596 0.002*

Troponin I NIDALSGMEGR[Dea]K (SEQ ID NO: 18) P19429 0.02

Troponin I ESLDLR[Dea]AHLK (SEQ ID NO: 19) PI 9429 0.02

Troponin I YR[Dea]AYATEPHAK (SEQ ID NO: 168) P19429 0.09

Troponin I [PGQ]-QELEREAEER[Dea]R[Dea]GEK (SEQ ID P19429 0.49

NO: 169)

Troponin T PR[Dea] SFMPNLVPPK (SEQ ID NO: 20) E7EPW4 0.01

Troponin T GERVDFDDIHR[Dea]K (SEQ ID NO: 170) E7EPW4 0.06

Troponin T EAEDGPMEESKPKPR[Dea] (SEQ ID NO: 171) E7EPW4 0.23

Troponin T AEAETEETR[Dea]AEEDEEEEEAK (SEQ ID NO: E7EPW4 0.66

172)

Tubulin alpha- IB chain HVPR[Dea]AVFVDLEPTVIDEVR[Dea]TGTYR[De P68363 0.43 a]QLFHPEQLITGK (SEQ ID NO: 173)

Tubulin alpha-4A chain HVPR[Dea]AVFVDLEPTVIDEIRN[Dea]GPYRQ[D P68366 0.26 ea]LFHPEQLITGK (SEQ ID NO: 174)

Tubulin alpha-4A chain EIIDPVLDR[Dea]IR[Dea]K (SEQ ID NO: 175) P68366 0.72

Tubulin alpha-8 chain ED AANNYAR[Dea] GHYTVGK (SEQ ID NO: 176) C9J2C0 0.37

Vimentin FADLSEAAN[Dea]RNNDALR[Dea]QAK (SEQ ID P08670 0.03

NO: 22)

Vimentin TVETR[Dea]DGQVINETSQHHDDLE (SEQ ID NO: P08670 0.09

177)

Heat shock protein beta-1 LATQSNEITIPVTFESR[Dea]AQLGGPEAAK (SEQ P04792 0.34

ID NO: 178)

Heat shock protein beta-7 RHPHTEH VQQTFR[Dea] TEIK (SEQ ID NO: 9) Q9UBY9 0.05

Leucine-rich PPR motif- IPENIYRGIR[Dea]N[Dea]LLESYHVPELIK (SEQ P42704 0.55 containing protein ID NO: 179)

Troponin C SEEELSDLFR[Dea]MFDK (SEQ ID NO: 180) P63316 0.69

Alpha- 1 -antitrypsin ELDR[Dea]DTVFALVN[Dea]YIFFK (SEQ ID NO: P01009 0.61

181)

Apolipoprotein A-I [PGE] -EN[Dea] GGAR[Dea]L AEYHAK (SEQ ID P02647 0.58

NO: 182)

Apolipoprotein A-I DSGR[Dea]DYVSQFEGSALGK (SEQ ID NO: 183) P02647 0.90

Apolipoprotein A-I AKPALEDLR[Dea]QGLLPVLESFK (SEQ ID NO: P02647 0.96

184)

Calreticulin PR[Dea]QIDNPDYK (SEQ ID NO: 185) P27797 0.17

Calumenin TEREQFVEFR[Dea]DK (SEQ ID NO: 186) 043852 0.06

Sarcalumenin IERpea]AITQELPGLLGSLGLGK (SEQ ID NO: Q86TD4 0.24

187)

Sarcalumenin TIEGIVMAADSAR[Dea] SFSPLEK (SEQ ID NO: Q86TD4 0.60

188)

Decorin ISR[Dea]VDAASLK (SEQ ID NO: 189) P07585 0.25 Protein Peptide UniProtKB p kw

Accessions

Beta-2 -glycoprotein 1 ATWYQGER[Dea]VK (SEQ ID NO: 190) P02749 0.55

Cathepsin D VER[Dea]QVFGEATK (SEQ ID NO: 191) P07339 0.14

Lumican YLYLR[Dea]NNQIDHIDEK (SEQ ID NO: 192) P51884 0.66

Myoglobin ALELFR[Dea]K (SEQ ID NO: 193) P02144 0.11

Ubiquitin carboxyl-terminal EEDPATGTGDPPR[Dea]K (SEQ ID NO: 194) P45974 0.50 hydrolase 5

2,4-dienoyl-CoA reductase GAFSR[Dea]LDPTGTFEK (SEQ ID NO: 195) B7Z6B8 0.26

28S ribosomal protein S36 PNVSEALR[Dea]SAGLPSHSSVISQHSK (SEQ ID P82909 0.29

NO: 196)

3-ketoacyl-CoA thiolase YALQSQQR[Dea]WK (SEQ ID NO: 197) P42765 0.55

3-ketoacyl-CoA thiolase VSPETVDSVIMGNVLQSSSDAIYLAR[Dea]HVGL P42765 0.82

R[Dea]VGIPK (SEQ ID NO: 198)

Acetyl-CoA acetyltransferase EVVIVSATR[Dea]TPIGSFLGSLSLLPATK (SEQ P24752 0.07

ID NO: 199)

Aconitate hydratase SYLR[Dea]LRPDRVAMQDAT[Dhy]AQ[Dea]M[Ox Q99798 0.01 i]AMLQFISSGLSK (SEQ ID NO: 25)

Aconitate hydratase, ANSVR[Dea]NAVTQEFGPVPDTAR[Dea]YYK Q99798 0.03

(SEQ ID NO: 26)

Aconitate hydratase, IVYGHLDDPASQEIER[Dea]GK (SEQ ID NO: 27) Q99798 0.03

Aconitate hydratase SYLRLR[Dea]PDRVAMQD ATAQMAMLQFIS SGL Q99798 0.14

SK (SEQ ID NO: 200)

Aconitate hydratase FR[Dea] GHLDNISNNLLIGAINIEN[Dea] GK (SEQ Q99798 0.19

ID NO: 201)

Acyl-Coenzyme A AFTGFIVEADTPGIQIGR[Dea]K (SEQ ID NO: 202) Q5T4U5 0.15 dehydrogenase, C-4 to C-12

straight chain, isoform

CRA a

Acyl-Coenzyme A FAR[Dea]EEIIPVAAEYDK (SEQ ID NO: 203) Q5T4U5 0.26 dehydrogenase, C-4 to C-12

straight chain, isoform

CRA a

Acyl-Coenzyme A TR[Dea]PWAAGAVGLAQRALDEATK (SEQ ID Q5T4U5 0.93 dehydrogenase, C-4 to C-12 NO: 204)

straight chain, isoform

CRA a

Adenylate kinase 4 LLR[Dea]AVILGPPGSGK (SEQ ID NO: 28) P27144 0.00

Adenylate kinase isoenzyme KR[Dea]GETSGR[Dea]VDDNEETIK (SEQ ID NO: Q5T9B7 0.95 1 205)

Alcohol dehydrogenase VAGASR[Dea]IIGVDINK (SEQ ID NO: 29) PI 1766 0.05 class-3

Aldehyde dehydrogenase VAFTGSTEIGR[Dea] VIQ VAAGS SNLK (SEQ ID P05091 0.08

NO: 206)

Aldehyde dehydrogenase SR[Dea] WGNPFDSK (SEQ ID NO: 207) P05091 0.55

Aspartate aminotransferase PYVLPSVR[Dea]K (SEQ ID NO: 208) P00505 0.22

ATP synthase subunit alpha R[Dea]TGAIVDVPVGEELLGR[Dea]WDALGNAI P25705 0.00

DGK (SEQ ID NO: 30)

ATP synthase subunit alpha AIEEQVAVIYAGVR[Dea] GYLDK (SEQ ID NO: P25705 0.01

31)

ATP synthase subunit alpha GIRP AINVGL S VSR[Dea] VGS AAQ [Dea] TRAMK P25705 0.01 Protein Peptide UniProtKB p kw

Accessions

(SEQ ID NO: 32)

ATP synthase subunit alpha QGQYSPMAIEEQVAVIYAGVR[Dea] GYLDK P25705 0.03

(SEQ ID NO: 33)

ATP synthase subunit beta IPVGPETLGRIMNVIGEPIDER[Dea] GPIK (SEQ ID P06576 0.26

NO: 209)

ATP synthase subunit beta IPVGPETLGR[Dea]IMNVIGEPIDER[Dea]GPIK P06576 0.51

(SEQ ID NO: 210)

ATP synthase subunit beta IQR[Dea]FLSQPFQVAEVFTGHMGK (SEQ ID NO: P06576 0.70

211)

ATP synthase subunit beta AHGGYSVFAGVGERTR[Dea]EGN[Dea]DLYHEM P06576 0.87

IESGVTNLK (SEQ ID NO: 212)

ATP synthase subunit e PRAEEER[Dea]R[Dea]IAAEEK (SEQ ID NO: 213) P56385 0.32

ATP synthase-coupling SGGPVDASSEYQQELER[Dea]ELFK (SEQ ID NO: P18859 0.00 factor 6, 34)

Chloride intracellular channel YR[Dea]NFDIPK (SEQ ID NO: 35) Q9Y696 0.04 protein 4

Citrate synthase SQLSAAVTALNSESN[Dea]FARAYAQGISR[Dea] B4DJV2 0.34

TK (SEQ ID NO: 214)

Citrate synthase T WGQITVDMMYGGMR[Dea] GMK (SEQ ID NO: B4DJV2 0.75

215)

Creatine kinase B-type LR[Dea]FPAEDEFPDLSAHNNHMAK (SEQ ID P12277 0.33

NO: 216)

Creatine kinase B-type E VFTR[Dea]FC [CAM] TGLTQIETLFK (SEQ ID P12277 0.82

NO: 217)

Creatine kinase S-type, C[CAM]TR[Dea]AER[Dea]R[Dea]EVENVAITALE P17540 0.31

GLK (SEQ ID NO: 218)

Creatine kinase S-type, TR[Dea]AER[Dea]R[Dea]EVENVAITALEGLK P17540 0.59

(SEQ ID NO: 219)

Creatine kinase S-type, EVREQ[Dea]PR[Dea]LFPPSADYPDLRK (SEQ ID P17540 0.72

NO: 220)

Cytochrome c EER[Dea]ADLIAYLK (SEQ ID NO: 36) C9JFR7 0.01

Cytochrome c KEER[Dea]ADLIAYLK (SEQ ID NO: 221) C9JFR7 0.01

Cytochrome c oxidase EIYPYVIQELR[Dea]PTLNELGISTPEELGLDKV P20674 0.07 subunit 5A, (SEQ ID NO: 222)

Cytochrome c oxidase SHGSQETDEEFDARWVTYFN[Dea]KPDIDAWEL P20674 0.18 subunit 5A, R[Dea]K (SEQ ID NO: 223)

Cytochrome c oxidase ASGGGVPTDEEQATGLER[Dea]EIMLAAK (SEQ P10606 0.49 subunit 5A, ID NO: 224)

Delta- 1 -pyrroline-5 - VLR[Dea]NAAGNFYINDK (SEQ ID NO: 37) P30038 0.00 carboxylate dehydrogenase

Delta- 1 -pyrroline-5 - AADMLSGPR[Dea]R[Dea]AEILAK (SEQ ID NO: P30038 0.01 carboxylate dehydrogenase 38)

Delta- 1 -pyrroline-5 - VANEPVLAFTQGSPER[Dea]DALQK (SEQ ID P30038 0.03 carboxylate dehydrogenase NO: 39)

Delta- 1 -pyrroline-5 - TVIQAEIDAAAELIDFFR[Dea]FNAK (SEQ ID NO: P30038 0.12 carboxylate dehydrogenase 225)

Dihydrolipoyl dehydrogenase NFQR[Dea]ILQK (SEQ ID NO: 226) P09622 0.87

Electron transfer flavoprotein VWSGGR[Dea] GLK (SEQ ID NO: 227) P13804 0.18 subunit alpha Protein Peptide UniProtKB p kw

Accessions

Ketimine reductase mu- PGAHINAVGASR[Dea]PDWR[Dea]ELDDELMK Q14894 0.49 crystallin (SEQ ID NO: 252)

L-lactate dehydrogenase A GEMMDLQHGSLFLR[Dea]TPK (SEQ ID NO: 253) P00338 0.23 chain

L-lactate dehydrogenase B HR[Dea]VIGSGC[CAM]NLDSAR[Dea]FR[Dea]YL P07195 0.87 chain MAEK (SEQ ID NO: 254)

L-lactate dehydrogenase B IVWTAGVRQQEGESR[Dea]LNLVQRN[Dea]VN P07195 0.87 chain VFK (SEQ ID NO: 255)

Lon protease homolog, LQQ[Dea]RLGR[Dea]EVEEK (SEQ ID NO: 256) K7EKE6 0.97

Malate dehydrogenase, GC[CAM]DVWIPAGVPR[Dea]K (SEQ ID NO: P40926 0.49

257)

Malate dehydrogenase, NSPLVSR[Dea]LTLYDIAHTPGVAADLSHIETK P40926 0.97

(SEQ ID NO: 258)

Methylcrotonoyl-CoA QEGIIFIGPPPSAIR[Dea]DMGIK (SEQ ID NO: 259) Q96RQ3 0.31 carboxylase subunit alpha,

Methylmalonate- NLR[Dea]VNAGDQPGADLGPLITPQAK (SEQ ID Q02252 0.70 semialdehyde dehydrogenase NO: 260)

[acylating],

NAD(P) transhydrogenase AATITPFR[Dea]K (SEQ ID NO: 43) Q13423 0.05

NAD(P) transhydrogenase, SMGAIVR[Dea]GFDTR[Dea]AAALEQFK (SEQ ID Q13423 0.11

NO: 261)

NADH dehydrogenase PIFSR[Dea]DMNEAK (SEQ ID NO: 262) P56556 0.16 [ubiquinone] 1 alpha

subcomplex subunit 6

Peroxiredoxin-1 R[Dea]TIAQDYGVLK (SEQ ID NO: 263) Q06830 0.21

Peroxiredoxin-2 EGGLGPLNIPLLADVTR[Dea]R[Dea]LSEDYGVL P32119 0.16

K (SEQ ID NO: 264)

Peroxiredoxin-5, VR[Dea]LLADPTGAFGK (SEQ ID NO: 265) P30044 0.16

Peroxiredoxin-6 LPFPIIDDR[Dea]NR[Dea]ELAILLGMLDPAEK P30041 0.68

(SEQ ID NO: 266)

Phosphate carrier protein, VYFR[Dea]LPRPPPPEMPESLK (SEQ ID NO: 44) Q00325 0.00

Pyruvate dehydrogenase El C[CAM]DLHR[Dea]LEEGPPVTTVLTR[Dea]EDGL P08559 0.01 component subunit alpha, K (SEQ ID NO: 45)

somatic form,

Pyruvate dehydrogenase El DR[Dea]MVNSNLASVEELK (SEQ ID NO: 267) P08559 0.60 component subunit alpha,

somatic form,

Pyruvate dehydrogenase HSLDASQGTATGPR[Dea]GIFTK (SEQ ID NO: 000330 0.37 protein X component, 268)

Short-chain specific acyl- SAAWFASTDR[Dea]ALQNK(SEQ ID NO: 269) P16219 0.54 CoA dehydrogenase,

Stress-70 protein, SDIGEVILVGGMTR[Dea]MPK (SEQ ID NO: 270) P38646 0.11

Stress-70 protein, IVR[Dea]ASN[Dea]GDAWVEAHGK (SEQ ID NO: P38646 0.16

271)

Succinate dehydrogenase VTLEYR[Dea]PVIDK (SEQ ID NO: 272) P31040 0.07 [ubiquinone] flavoprotein

subunit,

Succinate dehydrogenase IYQR[Dea]AFGGQSLK (SEQ ID NO: 273) P31040 0.40 [ubiquinone] flavoprotein

subunit,

Succinate dehydrogenase FAIYR[Dea]WDPDK (SEQ ID NO: 46) P21912 0.05 Protein Peptide UniProtKB p kw

Accessions

[ubiquinone] iron-sulfur

subunit,

Succinyl-CoA ligase TR[Dea]LIGPNC[CAM]PGVINPGEC[CAM]K (SEQ P53597 0.02 [ADP/GDP-forming] subunit ID NO: 47)

alpha,

Succinyl-CoA ligase PWSFIAGLTAPPGR[Dea]R[Dea]MGHAGAIIAG P53597 0.09 [ADP/GDP-forming] subunit GK (SEQ ID NO: 274)

alpha,

Succinyl-CoA ligase [ADP- INFD SNS AYR[Dea]QK (SEQ ID NO: 275) Q9P2R7 0.27 forming] subunit beta,

Succinyl-CoA ligase [ADP- GRIC[CAM]N[Dea]QVLVC[CAM]ER[Dea]K (SEQ Q9P2R7 0.39 forming] subunit beta, ID NO: 276)

Succinyl-CoA ligase [ADP- IFDLQDWTQEDER[Dea]DK (SEQ ID NO: 277) Q9P2R7 0.73 forming] subunit beta,

Succinyl-CoA:3-ketoacid ADR[Dea]AGNVTFR[Dea]K (SEQ ID NO: 48) P55809 0.04 coenzyme A transferase 1,

Succinyl-CoA:3-ketoacid GLTAVSNNAGVDNFGLGLLLR[Dea]SK (SEQ ID P55809 0.93 coenzyme A transferase 1, NO: 278)

Thioredoxin-dependent HLSVNDLPVGR[Dea] SVEETLRLVK (SEQ ID NO: P30048 0.18 peroxide reductase, 279)

Thioredoxin-dependent HLSVNDLPVGR[Dea] SVEETLR[Dea]LVK (SEQ P30048 0.68 peroxide reductase, ID NO: 280)

Trifunctional enzyme subunit ALTSFER[Dea]D SIFSNLTGQLDYQGFEK (SEQ P40939 0.99 alpha, ID NO: 281)

Very long-chain specific SDSHPSDALTR[Dea]K (SEQ ID NO: 282) P49748 0.32 acyl-CoA dehydrogenase,

Very long-chain specific ITAFWER[Dea]GFGGITHGPPEK (SEQ ID NO: P49748 0.55 acyl-CoA dehydrogenase, 283)

Very long-chain specific AL VER[Dea] GGWTSNPLGF (SEQ ID NO: 284) P49748 0.87 acyl-CoA dehydrogenase,

Voltage-dependent anion- SR[Dea]VTQSNFAVGYK (SEQ ID NO: 49) P21796 0.02 selective channel protein 1

60 kDa heat shock protein, FDR[Dea] GYISPYFINTSK (SEQ ID NO: 50) P10809 0.04

61 kDa heat shock protein, WR[Dea]TALLDAAGVASLLTTAEVWTEIPK P10809 0.07

(SEQ ID NO: 285)

62 kDa heat shock protein, LVQD VANNTNEEAGDGTTTATVLAR[Dea] SIAK P10809 0.14

(SEQ ID NO: 286)

63 kDa heat shock protein, GANPVEIR[Dea]R[Dea] GVML AVD AVIAELK P10809 0.22

(SEQ ID NO: 287)

64 kDa heat shock protein, GR[Dea]TVIIEQSWGSPK (SEQ ID NO: 288) P10809 0.55

65 kDa heat shock protein, GANPVEIRR[Dea]GVMLAVD AVIAELK (SEQ ID P10809 0.58

NO: 289)

66 kDa heat shock protein, FGADAR[Dea]ALMLQGVDLLADAVAVTMGPK P10809 0.93

(SEQ ID NO: 290)

Cytoplasmic aconitate PAR[Dea]VILQDFTGVPAWDFAAMR[Dea]DAV P21399 0.77 hydratase K (SEQ ID NO: 291)

Polymerase I and transcript TVRGSLER[Dea]QAGQIK (SEQ ID NO: 292) Q6NZI2 0.09 release factor

Polymerase I and transcript TVR[Dea]GSLER[Dea]QAGQIK (SEQ ID NO: 293) Q6NZI2 0.34 release factor

Trifunctional enzyme subunit PNIR[Dea]NWWDGVR[Dea]TPFLLSGTSYK P55084 0.02 beta, (SEQ ID NO: 51) Protein Peptide UniProtKB p kw

Accessions

Aconitate hydratase, TGR[Dea]EDIANLADEFK (SEQ ID NO: 52) Q99798 0.00

Aconitate hydratase, R[Dea]LQLLEPFDK (SEQ ID NO: 53) Q99798 0.01

Dual specificity protein LGITHVLNAAEGR[Dea] SFMHVNTNANFYK P51452 0.01 phosphatase 3 (SEQ ID NO: 54)

Elongation factor 1 -alpha 2 ASGVSLLEALDTILPPTR[Dea]PTDK (SEQ ID NO: Q05639 0.45

294)

Elongation factor 1 -alpha 2 IGGIGTVPVGR[Dea]VETGILR[Dea]PGMWTFAP Q05639 0.80

VNITTEVK (SEQ ID NO: 295)

Elongation factor 2 VNFTVDQIR[Dea]AIMDK (SEQ ID NO: 296) P13639 0.09

Eukaryotic translation VAPAQPSEEGPGR[Dea]K (SEQ ID NO: 297) E7EX17 0.95 initiation factor 4B

Histone H2B type 1-M AVR[Dea]LLLPGELAK (SEQ ID NO: 298) Q99879 0.99

Histone H3 (Fragment) LPFQR[Dea]LVR[Dea]EIAQDFK (SEQ ID NO: K7EK07 0.36

299)

Histone H4 RISGLIYEETR[Dea] GVLK (SEQ ID NO: 300) P62805 0.65

Histone H4 VFLENVIR[Dea]DAVTYTEHAK (SEQ ID NO: P62805 0.74

301)

Neuroblast differentiation- FGVSTGR[Dea]EGQTPK (SEQ ID NO: 55) Q09666 0.01 associated protein AHNAK

Fibrinogen alpha chain ESS SHHPGI AEFPSR[Dea] GK (SEQ ID NO: 302) P02671 0.39

Fibrinogen gamma chain R[Dea]LDGSVDFK (SEQ ID NO: 303) C9JEU5 0.62

Haptoglobin LR[Dea]TEGDGVYTLNNEK (SEQ ID NO: 304) P00738 0.40

Serotransferrin DYELLC[CAM]LDGTR[Dea]K (SEQ ID NO: 305) P02787 0.10

Collagen alpha-3(VI) chain D WFLLDGSEGVR[Dea] SGFPLLK (SEQ ID NO: E7ENL6 0.03

56)

[0112] Table 1C: Citrullinated peptides that are upregulated or downregulated in healthy versus ischemic and healthy versus IDCM subjects. I: Ischemic Heart Disease (ISHD); I/N = ischemia vs. normal (healthy); ID= Idiopathic Cardiomyopathy (IDCM); ID/N = IDCM vs. control (healthy).

Protein I/N I

Peptidyl-prolyl cis-trans isomerase A 4.691862761 up

Delta-l-pyrroline-5-carboxylate dehydrogenase 4.531765219 up

Chloride intracellular channel protein 4 4.143020683 up

Alcohol dehydrogenase IB 3.979284116 up

Adenylate kinase 4 3.954001114 up

Fatty acid-binding protein 3.891772332 up

Myosin-binding protein C 3.770886194 up

Aconitate hydratase 3.740779581 up

Aconitate hydratase,\ 3.54332942 up

Delta-l-pyrroline-5-carboxylate dehydrogenase 3.484011583 up

Dual specificity protein phosphatase 3 3.348178139 up

Pyruvate dehydrogenase El component subunit alpha, somatic form 3.336292196 up

Voltage-dependent anion-selective channel protein 1 3.075867039 up

Heat shock protein HSP 90-alpha 3.018162117 up

Succinate dehydrogenase [ubiquinone] iron-sulfur subunit 2.797967329 up

Aconitate hydratase 2.646902712 up

Carbonic anhydrase 3 2.393105549 up

Serum deprivation-response protein 2.181113303 up

ATP synthase subunit alpha 2.125964715 up

Succinyl-CoA:3-ketoacid coenzyme A transferase 1 2.02609809 up

Alcohol dehydrogenase class-3 1.955112799 up

Succinyl-CoA ligase [ADP/GDP-forming] subunit alpha 1.813434217 up

Troponin I, cardiac muscle 1.770629108 up

ATP synthase subunit alpha 1.757665967 up

ATP synthase subunit alpha 1.716317069 up

Filamin-C 1.707322518 up

60 kDa heat shock protein 1.691322503 up

Heat shock protein beta-7 1.659329245 up

Myozenin-2 1.640224333 up

Cytochrome c (Fragment) 1.516000483 up

LIM domain-binding protein 3 1.456615429 up

Trifunctional enzyme subunit beta 1.262914035 up

Phosphate carrier protein 0.90684035 down

ATP synthase subunit alpha 0.821046528 down

Collagen alpha-3(VI) chain 0.787448747 down

Myozenin-2 0.750016161 down

Troponin I 0.707050891 down

NAD(P) transhydrogenase 0.689499324 down

Alpha-crystallin B chain 0.574071929 down

ATP synthase-coupling factor 6 0.558912444 down Protein I/N I

Troponin T, cardiac muscle (Fragment) 0.474388613 down

Beta-enolase 0.324050919 down

Adenylate kinase isoenzyme 1 0.134886108 down

[0113] Table ID: Citrullinated peptides that are upregulated or downregulated in healthy versus ischemic and healthy versus IDCM subjects. I: Ischemic Heart Disease (ISHD); I/N = ischemia vs. normal (healthy); ID= Idiopathic Cardiomyopathy (IDCM); ID/N = IDCM vs. control (healthy)

Protein ID/N ID

Succinate dehydrogenase [ubiquinone] iron-sulfur subunit 1.182430989 up

Dual specificity protein phosphatase 3 1.089131195 up

Serum deprivation-response protein 1.05734493 up

60 kDa heat shock protein 0.676500721 down

Heat shock protein beta-7 0.569844647 down

Filamin-C 0.543903731 down

Alcohol dehydrogenase class-3 0.539343446 down

Adenylate kinase 4 0.516646378 down

Fatty acid-binding protein 0.500998082 down

Myozenin-2 0.483486835 down

Neuroblast differentiation-associated protein AHNAK 0.440092248 down

Cytochrome c (Fragment) 0.256014197 down

Succinyl-CoA ligase [ADP/GDP-forming] subunit alpha 0.234602593 down

Peptidyl-prolyl cis-trans isomerase A 0.231427358 down

Carbonic anhydrase 3 0.230059909 down

LIM domain-binding protein 3 0.21853459 down

Troponin I 0.188118204 down

Troponin I 0.184373163 down

Troponin T 0.153543871 down

Alpha-crystallin B chain 0.145172906 down

Succinyl-CoA:3-ketoacid coenzyme A 0.121733077 down transferase 1

ATP synthase-coupling factor 6 0.076789945 down

Adenylate kinase isoenzyme 1 0.023786955 down

Beta-enolase 0.013231973 down

[0114] Table IE: Proteins that were up in both I and ID groups. I: Ischemic Heart Disease (ISHD); I/N = ischemia vs. normal (healthy); ID= Idiopathic Cardiomyopathy (IDCM); ID/N = IDCM vs. control (healthy).

Protein I/N I ID/N ID

Aconitate hydratase 3.54332942 up 3.868674325 up

Pyruvate dehydrogenase El component subunit 3.336292196 up 3.241986763 up alpha, somatic form

Aconitate hydratase 8.871956482 up 3.202560876 up

Vimentin 6.886890573 up 3.18977634 up

Trifunctional enzyme subunit beta 1.262914035 up 3.078371104 up

Aconitate hydratase 5.18248378 up 3.033531051 up

Myosin-binding protein C 3.770886194 up 2.938608028 up

Chloride intracellular channel protein 4 4.143020683 up 2.768865949 up

Aconitate hydratase 3.740779581 up 2.733376766 up

Heat shock protein HSP 90-alpha 3.018162117 up 2.673523729 up

Delta- 1 -py rroline-5 -carboxy late dehydrogenase 7.552745345 up 2.570795078 up

Serum deprivation-response protein 2.181113303 up 2.396582653 up

Delta- 1 -py rroline-5 -carboxy late dehydrogenase 3.484011583 up 2.194292783 up

Alcohol dehydrogenase IB 3.979284116 up 1.513050932 up

Aconitate hydratase 2.646902712 up 1.359569141 up

Succinate dehydrogenase [ubiquinone] iron-sulfur 2.797967329 up 1.182430989 up subunit

Dual specificity protein phosphatase 3 3.348178139 up 1.089131195 up

Serum deprivation-response protein 5.310526396 up 1.05734493 up

[0115] Table IF: Proteins that were down in both I and ID groups. I: Ischemic Heart Disease (ISHD); I/N = ischemia vs. normal (healthy); ID= Idiopathic Cardiomyopathy (IDCM); ID/N = IDCM vs. control (healthy).

[0116] Western blotting of myofilament- and cytosolic-enriched fractions obtained from ISHD, IDCM and non-failing donor hearts (n=10/group) using an anti -modified citrulline antibody confirmed that citrullination occurs to intracellular proteins. Although there was no difference (Figure IB ) in the overall immunoreactivity between groups with regard to the number of bands or band density per blot, in-gel digestion and subsequent mass spectrometry of the immuno-reactive bands identified the major sarcomeric proteins at their expected molecular weight (e.g. myosin heavy and light chains and actin. It must be noted that quantitative assessment by immuno-lDE was confounded by the presence of other proteins in the gel bands and challenges of direct site-specific assessment using mass spectrometry.

[0117] To further validate citrullination of the high abundant sarcomeric proteins, DIGE 2D gel electrophoresis (pH 4-7, 10% SDS PAGE) was carried out. Myofilament- and cytosolic- enriched fractions of ISHD, IDCM and non-failing donor hearts (n=4/group) were pretreated with PAD2 to induce maximum citrullination, combined at a 1 : 1 ratio with the matching untreated samples and simultaneously resolved by 2D gel electrophoresis. The sarcomeric proteins, including actin, TM and myosin light chains were shown to be citrullinated with ectopic treatment of PAD2 (Figure 2).

Biochemical assessment of modified sarcomeric proteins.

[0118] To test whether citrullination can affect sarcomeric protein function, actin, FDVIM, TM and troponin were citrullinated by ectopic PAD2 and then compared to respective unmodified recombinant proteins to determine if citrullination affects their biochemical, structural, or enzymatic properties. The binding of citrullinated or untreated FDVIM, TM or TM-Tnl to F- actin was determined using classical co-sedimentation assays (Figure 3 A-C). Citrullination of actin did not alter F-actin formation and over 95% of the actin was pelleted upon centrifugation. Binding of citrullinated TM to F-actin was enhanced compared to unmodified TM (Figure 3A). Since TM and FDVIM can affect binding of each other to F-actin in a cooperative manner [Galinska A et al. The C terminus of cardiac troponin I stabilizes the Ca2+-activated state of tropomyosin on actin filaments. Circ Res 2010, 106:705-711], we tested for cooperativity under conditions in which binding of TM alone to F-actin is poor, but increased upon the binding of myosin heads to F-actin [Eaton BL. Tropomyosin binding to F- actin induced by myosin heads. Science 1976, 192: 1337-1339]. As illustrated in Figure 3B, at low salt concentration citrullinated TM in the presence of FDVIM was able to bind to F-actin.

[0119] The binding of cardiac Tnl to F-actin in the presence of TM was performed by co- sedimenting citrullinated Tnl in the presence of F-actin and TM. It was found that both citrullinated and non-citrullinated forms of cardiac Tnl bound to F-actin equally well (Figure 3C) Inhibition of HMM-ATPase activity.

[0120] To verify a possible modulation of the actomyosin HMM-ATPase activity by citrullination of sarcomeric proteins, experiments were performed in the presence of citrullinated HMM, F-actin and TM. The data summarized in Figure 4A show that, in the presence of citrullinated HMM (0.5μιηο1/Ι. HMM and 7μιηο1/Ι. F-actin), the actomyosin HMM-ATPase activity decreased from 0.32 ± 0.01 nmol Pi to 0.22 ± 0.002 nmol Pi, corresponding to 30% decrease in the enzyme activity. In contrast, citrullination of F-actin enhanced actomyosin HMM-ATPase activity up to 55% on the enzyme activity (Figure 4A). When both F-actin and HMM were citrullinated, the rate of ATP hydrolysis remained greater than control (0.368± 0.005 nmol Pi to 0.32 ± 0.01 nmol Pi). Figure 4B showed that actomyosin HMM-ATPase activity was affected by TM citrullination. The inhibition of actomyosin HMM-ATPase conferred by non-citrullinated and citrullinated Tnl was compared. Addition of non-citrullinated Tnl to HMM-F-actin-TM caused a decrease in the ATPase rate at 37 °C. Citrullinated Tnl acted similar to non-citrullinated Tnl and caused a decrease in the ATPase with no significant deference to non-citrullinated form.

PA 1)2 reduces calcium sensitivity in skinned myocytes

[01211 Chemically skinned cardiomyocytes isolated from the left ventricle of wild-type C57B16 male mice were exposed to varying concentrations of calcium (n=8 myocytes from 3 mice per group. Figure 4C-F). PAD2 treatment had no effect either maximal calcium activated force (Fma _X ) or hill coefficient (nil). However, PAD2 caused a rightward shift in the force-calcium relationship that indicates an increase in EC ₅₀ , or a decrease in calcium- sensitivity (p=0.009, Figure 4C). This suggests that citrullination of myofilament proteins causes a loss-of- function phenotype, reducing its ability to generate force in response to intracellular calcium.

Analysis of PAD mRNA expression.

[0122] To determine the cell specificity of the PAD isoform expression in the myocardium, mRNA expression level was determined using isoform specific primers by Nested-PCR on isolated cardiac myocytes and fibroblasts (Figure 5). PAD2 was the primary isoform expressed in both cardiomyocytes and cardiac fibroblasts, although fibroblasts also had significant expression of PAD1 and PAD4 mRNA. In contrast, PAD3 mRNA was not detected in any of the cell types and conditions tested (Figure 5, note no band at 200 bp, see online supplement for more details).

Example 3

[0123] Our experimental findings characterize citrullinated proteins in the normal and HF myocardium. The analysis revealed that citrullination is enriched in the mitochondria and sarcomere subproteomes. Protein citrullination has broad cellular distribution (Figure 1) but is highly enriched in the mitochondria and sacromeric subproteome. A close connection between these two subproteomes is not unexpected due to the high energetic requirements of the sarcomere. We speculated that citrullination, similar to phosphorylation and acetylation, could potentially regulate muscle-contractile proteins in a coordinated manner. Interestingly, the multiple enzymes involved in metabolic pathways/metabolism were up regulated in ischemia but down regulated in IDCM (Figure 6). To understand the topology and functional annotation of citrullinated protein-protein interaction in the heart system the protein network was constructed using the STRING database (Figure 2B). The entire protein network consisted of high scoring interaction partners (STRING relevant confidence score > 0.5, ischemia/IDCM vs. control). The visual analysis of this sub-network showed that citrullinated proteins are interacted to each other and are involved in the metabolism and respiratory chain targets, contraction and signal transduction systems. As an initial assessment to understand potential functional consequences of the citrullination, recombinant sarcomere proteins FDVIM, F-actin, TM and Tnl were used as model proteins to study the interactions that govern the thick and thin filament function and by measured the contractile properties of single skinned myocytes treated with PAD2.

[0124] Ca ²⁺ -dependent alterations to Tn alter an azimuthal movement of TM on the actin surface, which allows myosin binding and cross-bridge isomerization to strong binding, force-producing states and muscle contraction. At low intracellular Ca ²⁺ , Tn-TM sterically blocks myosin binding sites on actin (blocked state), while in presence of elevated Ca ²⁺ Tn- TM moves and partially exposes the myosin binding sites on F-actin (closed state). Myosin binding in the presence of Ca ²⁺ is required for full activation. It is a combination of Ca ²⁺ - induced Tnl conformational change which is, in part, transmitted via TnT to TM (especially the Tl region that binds along TM) as well as myosin binding that influence the exact positioning of TM on the actin filament. Based on our MS data, citrullination sites on myosin, actin, TM, Tnl and TnT are in regions what can influence these interactions and thus, the actomyosin HMM-ATPase activity and contraction. Below outlines the potential impact based on the biochemical and physiological experiments presented in this manuscript.

[0125] First, intrinsic actomyosin HMM-ATPase was inhibited by citrullination, but this was overcome in the presence of F-actin regardless of whether F-actin is citrullinated or not. Citrullinated F-actin was a more potent modulator of FEVEVI enzymatic activity and increased actomyosin FIMM-ATPase rate by 55% compared to the unmodified F-actin. This suggests that citrullination of F-actin changes the confirmation of the actin filament to alter the ease of ATP hydrolysis by the myosin once it is bound. The actin-FEVEVI interaction was also affected by the citrullination of TM. Citrullinated TM displayed enhanced binding to F-actin compared to unmodified TM. This was also observed in the presence of FEVIM (based on the centrifugation assays). This correlated to an inhibition of the citrullinated TM-actin-FEVEVI ATPase activity compared to the non-citrullinated TM, suggesting that citrullinated TM altered the ability of FEVEVI to bind to the actin filament (Figure 7). Intriguingly, citrullinated Tnl, like the unmodified protein complex, bound tightly to actin-TM based on centrifugation studies. Figure 3C shows that >95% of the unmodified and modified Tnl was present in the pellet.

[0126] Biochemical results were related to our physiological finding and showed that citrullination of the sarcomeric proteins caused a decrease in Ca ²⁺ -sensitivity in the skinned cardiac myocyte (Figure 4C). On Tnl, C-terminus residues 191 to 210, which contains the citrullinated residue 203, is primarily responsible for maintaining the TM conformation that prevents cross bridge cycling. Thus, not only does Tnl promote the blocked state, but also contributes to the stabilization of TM in the closed state. In mice, cardiac Tnl containing cardiomyopathy mutations, R192H or R204H (which are equivalent to the human sequence of 191 and 203 the latter being the same citrullinatable residue as discussed above) increases the binding affinity of Tn for actin-TM. Perhaps this is augmented by the citrullination of TnT at residue 77, which is located in the N-terminal tail (TnTl) that lies along TM and has been shown to be involved in cooperativity of the actin-TM-Tn filament. In addition, binding of Ca ²⁺ or myosin to actin-TM-Tn can displace Tnl residues 151 and 188, which flank the second citrullinated residue 169, away from the outer domain of F-actin. This is consistent with an azimuthal displacement to Tnl-TM by Ca ²⁺ that can expose the high affinity-binding site on F- actin for myosin. Studies on familial hypertrophic cardiomyopathic mutations occurring on thick filament proteins (TM, MHC, MyBP-C, ELC, and RLC) show that a change in one amino acid side chain can have an enormous effect on cardiac morphology and function. Furthermore, factors that lead to abnormal contraction and relaxation in the failing heart include metabolic pathway abnormalities that result in decreased energy production, energy transfer and energy utilization.

[0127] Finally, to better understand the involvement of citrullinated proteins in the heart, it is important to obtain insight about cell specificity. Previous data acquired by the immunohistochemistry showed that PADs 1-3 and to some degree, PAD6 were detected in cardiomyocytes with PADs 2 and 4 found in endothelial cells and vascular smooth muscle cells. In the present study, we relied not on antibodies and tissue slices, but rather examined the mRNA expression of PAD family members in isolated mouse cardiomyocytes and cardiac fibroblasts. The data showed that PAD2 was primarily expressed in both cardiomyocytes and cardiac fibroblasts whereas PAD1 and PAD4 mRNA were the major forms in the cardiac fibroblasts.

[0128] We have presented previously unexplored roles for citrullination in the heart. Ultimately, identification of citrullination of the majority of the sarcomeric proteins and alterations in their biochemical properties suggest that there is potentially a new PTM regulation of cardiac contractility. Since citrullination at some of these residues were increased in the myocardium of individuals with HF compared to the controls, suggest that citrullination could play a role, in the decrease contractile dysfunction in FIF.

Example 4: Citrullinated peptides in neurodegenerative diseases

[0129] Citrullination is a PTM of arginine residues that is characterized by the oxidization of the side chain. This modification is catalyzed by Peptidyl Arginine Deiminase enzymes (PAD) that causes the conversion of arginine to citrulline. Citrullination, also known as deimination occurs when there is a loss of a positive charge and reduction in hydrogen- bonding ability, ultimately causing a change in the protein's chemical structure. This modification plays both a physiological role, for instance during apoptosis and epigenetics, and a pathological role in cancer or diseases of the central nervous system [3]. Furthermore, anti-citrullinated protein antibodies (ACPAs) are present in ~60% of patients with rheumatoid arthritis (RA) and are associated with aggressive disease and a poor prognosis [4, 5]. Genetic studies of RA have identified many disease-prone SNPs at >100 non-HLA gene loci [6]. Therefore, the investigation to associate citrullination, SNPs and disease can provide an insight to many pathological disorders. Protocol overview

[0130] The purpose of this protocol is to build citrullinated assay libraries that support accurate detection of citrullinated proteins/peptides in vivo. The protocol covers the sample preparation, the acquisition of high-quality fragment ion spectra in DIA mode, the spectral citrullinated library generation and bioinformatics pipeline for binning the citrullinated peptides based on quality. Furthermore, additional tools were developed to translate the large datasets into knowledge by further annotating the peptide data with known vs. novel, and linkage to disease associated genomic alteration information.

Materials

[0131] Reagents: Rabbit skeletal muscle PAD cocktail (PAD) (SignalChem); Sequencing grade Lys-C protease (WAKO); iRT retention time peptides [13]; FASP™ Protein Digestion Kit (Expedeon),

[0132] Human brain tissue. Healthy control post-mortem human brain tissues corresponding to superior frontal gyrus (SFG) (n=10) obtained from NTH NeuroBioBank (NBB). The cases had neither a clinical history nor a neuropathology diagnosis of neurologic disease. Human ALS motor cortex(n=l) and ALS thoracic spinal cord (n=4) with corresponding healthy samples obtained from occipital lobe (n=3). Mouse brains: Alzheimer's model (1. 9 months old, lots of beta-amlyloid, memory deficitis and onset of neurodegeneration, n=5; 2. 9 months old, normal/healthy mouse, with inctact memory, n=5; 3.2 months old, little to no detectable beta-amyloid and intact memory, n=2; 4. 2 months old, healthy mouse, n=2 ). Stroke mouse (a middle cerebral artery occlusion stroke, n=5 Left side of brain where storoke ocured, right side of brain, corresponding healthy control); Healthy mouse (n=5, brain, aorta, skeletal muscle)..

[0133] Brain buffer A: Ice-cold low salt buffer: lOmM Tris, pH 7.5, 5mM EDTA, ImM DTT, 10% sucrose, lOmM b-glycerophosphate, lOmM sodium orthovanadate, lOmM tetrasodium pyrophosphate, 50mM sodium fluoride, 1 x Roche complete protease inhibitor cocktail).

[0134] Brain buffer B: 1% Triton, lOmM Tris, pH 7.5, 5mM EDTA, ImM DTT, 10% sucrose, lOmM b-glycerophosphate, lOmM sodium orthovanadate, lOmM tetrasodium pyrophosphate, 50mM sodium fluoride, 1 ^χ Roche complete protease inhibitor cocktail

[0135] PAD Buffer: 0.1M tris-HCl, pH7.4, 10 mM CaC12, 5mM DTT, lOug/ul aprotonin, 10μg/ul leupeptin, lOug/ul pepstatin. [0136] Equipment: High Speed Mixer Mill(MM400) (Retsch); Mixer Mill jar (# 22.008.0005 Retsch) and 7mm stainless steel ball (#22.455.0008 Retsch); Refrigenerated Bench-top centrifuge (Eppendorf 5415R), temperature 4°C and 20°C; Beckman Coulter Optima Ultracentrifuge with TLA 100.2 rotor; TripleTof 5600+ mass spectrometer (AB Sciex); TripleTof 6600+ mass spectrometer (AB Sciex); iProXpress (http://proteininformationresource.org/iproxpress2) [14]; Computer: PC with Microsoft Windows 7 (Microsoft), >4 GB of RAM, sufficient hard disk space (for the protocol case study >40 GB).

[0137] Software: Microsoft Windows 7 (Microsoft); MS Excel (Microsoft); Cytoscape Plugin BiNGO A Biological Network Gene Ontology Tool (http://baderlab.org/Software/EnrichmentMap/BingoTutorial).

[0138] Equipment Setup: Detailed instructions, including screenshots, for the installation of each software module are provided in Supplementary material.

[0139] Methods: 1. Brain sample preparation. TIMING 90 min to a few days (depends on number of samples); 2. Weigh tissue and record wet tissue weight. 3. Cut the tissue into small pieces with a scalpel or a tissue slice blade. 4. Transfer the tissue pieces into 3 volumes (w/v) of ice cold Buffer A. 5. Homogenize the tissue on ice using a homogenizer (30 s at 20 Hz using a single 7mm stainless-steel ball). 6. Centrifuge the sample for 15 min, 30800 rpm, 4°C using a TLA 100.2 rotor in a Beckman Coulter Optima Ultracentrifuge. 7. Transfer the supernatant into a clean tube. 8. To remaining pellet add buffer B (1 :3 tissue to buffer ratio) and homogenized at 30 Hz for 30 s in a cold Mixer Mill MM400. 9. Centrifuge at 100,000 rpm for 20 min in a TLA- 100.2 rotor (Beckman). 10. Transfer the supernatant into a clean tube and determine the protein concentration by the Bradford method.

[0140] Protein citrullination: Peptides can only be identified by targeted MS if they are included in the assay library. To cover proteins that are expressed in specific biological conditions we needed to prepare the pool of citrullinated peptides that theoretically represents the entire biological space. Most PAD isoforms are exclusively expressed in the cytoplasm, except for PAD4, which contains a nuclear localization signal and is indeed found to reside at least in part in the nucleus. It would therefore be reasonable to assume that the substrate specificity differs between the PAD isotypes [15, 16]. A key step in the process is the treatment of pooled tissue/sample(s) in vitro with a PAD cocktail to induce hypo- citrullination of accessible arginine residues. (1) Pool samples in each fraction in order to obtain 200 ug sample pool. (2) Mix up to 30μ1 of a protein pool with 150μ1 of PAD Buffer. (3) Add PAD cocktail (Catalog #P312-37C) in an enzyme/substrate (w/w) ratio of 1 :25, and incubate the reaction for 2 h at 37C, gentle shacking. Stop citrullination by adding EDTA until 50 mM final concentration. (4) Prepare the negative control the same way but add water instead of PAD cocktail. (5) Keep on ice until use.

[0141] Protein digestion. Protein Discovery's FASP Protein Digestion Kit was used in order to digest all samples with LysC in an enzyme/substrate (w/w) ratio of 1 :30, and incubate the reaction for 20h at 37°C, gentle shaking.

[0142] SWATH-MS Data Acquisition. Generation of SWATH-MS maps: Triple-TOF MS analysis in DDA and SWATH mode. A TripleTOF 6600 mass spectrometer (Sciex) was used for both data dependent acquisition to build peptide spectral ion library and SWATH- MS (data independent acquisition) for each individual sample analysis. The raw data was searched with ProteinPilot™ Software 5.0 to create a spectral ion library. Individual SWATH-MS runs were matched against the spectral library created in the presence or absence of PAD (plus and minus PAD).

[0143] Extensive identification and analysis of conserved citrullinated peptides. The blast on the peptide level was performed to find human homology (sequence-similar) equivalent of the mouse data obtained from brain stroke mouse model, Alzheirmer's and healthy mouse brain, as well as from mouse aorta and mosue skeletal muscle. The matching list only includes the top blastp-short hit, meaning >80% of peptide homology between mouse and human was found. In most cases, if the top hit was an isoform, the other isoforms and canonical protein was also conserved. Figure 9 shows the overview bioinformatics workflow for processing citrullinated spectra library file. Two different types of tissue sample, heart and brain, were used to develop the presented methodologies. The final SWATH assay library generated from the human brain consists of 360,202 peptides (16,275 unique). The bioinformatics pipeline extracted 1,530 unique peptides that were considered to be high confident and valid citrullinated peptides (Table 11). An additional 1,571 unique peptides were identified as potentially high confident valid citrullinated peptides, and the other remaining peptides were considered to be low confident (Table 11). It is important to note that a peptide may have more than 1 citrullinated R, and some peptides share the exact citrullinated R site on the full length protein. Therefore, the number of unique peptides does not equal the number of unique citrullinated sites. The total number of unique citrullinated sites (on full length protein) in Outputl, 2, and 3 is 2413.

[0144] Table 11. Summary of output files for brain sample plus and minus PAD treatment for brain samples (confidence threshold used was 0.995) (JF_150402_560O-plus/minusPAD).

Total Peptides [ Tots! Unique Peptides minus PAD plus/minus PAD minus PAD plus/minus PA dei - Smin

Not checked I..63? 36,202 S8 1,530 conf 0.9S

delta 5 min OR

conf <0.S5 OS no ass ¾238 30,382 1S2 .1,571 matched control

delta < 5 mi n OR

«;ηί<0.95 OR no fail 370 19,450 55 1,006 matched control

every line

Net checked 375,717 431,816 17.147 20.702 without Rf Dea

[0145] The citrullinated peptides are binned into 4 different output files based. Output 1 contains the high confidence peptides, which have a > 5min delta in retention time between the modified peptide and the unmodified peptide, plus a confidence score (user input). Output 2 and 3 contains peptides that had a citrullinated R residue; however, no unmodified peptide was found in the dataset limiting the ability to calculate the retention time difference. However, for Output 2 an N/Q residue is not within 5 amino acids on either side of the citrullinated R residue; whereas in Output 3 an N/Q residue is located within 5 amino acids on either side the citrulinated R residue. Output 4 contains all peptides in the original file that did not meet the criteria for Outputs 1-3.

[0146] Herein we describe a step-by-step protocol and an integrated, openly accessible computational pipeline to generate high-quality assay libraries for analysis of citrullinated peptides in complex biological samples. Using the high resolution of the time-of-flight mass analyzer (>30,000) that allowing for confident identification of the peptide elution profile and discrimination between near isobaric parent mass tolerance and bioinformatics pipeline that rules the outcomes we were able to identified citrullinated peptides over a dynamic range of 6 orders of magnitude and quantified those with relative abundance.

[0147] There are some chemical, proteomic and antibody-based procedures that are currently available for the analysis of citrullination in complex samples [15-17]. However, with the current methods it remains challenging to rapidly, consistently, reproducibly, accurately, and sensitively detect and quantify citrullinated peptides across multiple samples. Here we present a new strategy that systematically queries sample sets for the presence and quantity of citrullinated proteins with exact site of modified residues. It consists of using the information available in fragment ion spectral libraries, like relative intensities, chromatographic concurrence to mine the complete citrullinated fragment ion maps generated using DIA method.

[0148] In essence, our tools identify peak groups that uniquely associate with the targeted citrullinated peptide within the comprehensive SWATH MS signal map, and then compute a probability that the targeted citrullinated peptide has been correctly identified by following the set up rules in bioinformatics pipeline. In order to increase sensitivity a high-quality library of citrullinated assays is a prerequisite for SWATH MS and similar targeting MS methods. Ideally, the citrullinated peptides in the assay library cover all citrullinated proteins of interest for a particular study, or even an entire proteome. In order to make sure it is true samples on based the library is built on are in vitro citrullinated by PAD cocktail. This step helps improve detection of citrullinated proteins present in lower amounts as well as cover potentially new sites.

[0149] We show that SWATH™-MS can be successfully adopted for PTM analysis. Bioinformatics pipeline added for analysis of citrullinated peptide makes the analysis reliable, robust and quick. We believe that this methodology will increase popularity of analysis of citrullination in the field and shortly will be invaluable to elucidate the importance of this posttranslational modification in vivo.

[0150] As described herein, citrullination, the post-translational (PTM) conversion of arginine to citrulline by the family of peptidylarginine deiminase (PADs), has been commonly implicated as abnormal pathological features in neurodegenerative diseases such as prion diseases, multiple sclerosis, and Alzheimer's disease. This PTM could be a target for novel diagnostic or therapeutic agents. Therefore, an unambiguous and efficient method to identify citrullinated proteins and their modified residues is of extreme importance.

[0151] ALS, or amyotrophic lateral sclerosis, is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. The inventors investigated the role of citrullination in ALS disorders, with a focus on identifying a potentially clinically relevant PTM biomarker(s). [0152] Fig. 8B depicts construction of citrulinated ion library. Figure 8C depicts delta RT for R[Dea] and N/Q[Dea] compared to unmodified sequence. Respectively, peptide containing citrullinated Arg eluted later compared to unmodified peptides (>5 min difference). The same trend was observed between peptides with citrullinated R and peptides with N and Q deamination.

[0153] As shown in Fig. 8F, alteration in citrullination of any one or more of Glial fibrillary acidic protein, Glucose-6-phosphate isomerase, Vimentin, Endophilin-Al, Myelin basic protein, Delta-l-pyrroline-5-carboxylate dehydrogenase, Peroxiredoxin-5, Neurogranin, Dual specificity mitogen-activated protein kinase kinase 1, Tubulin polymerization- promoting protein family member 3, Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, Cysteine and glycine-rich protein 1, Cellular retinoic acid-binding protein 1 GMP reductase 2 and/or Guanine nucleotide-binding protein G(I)/G(S)/G(0) subunit gamma-3 may be indicative of ALS.

[0154] Table 2: Citrullinated peptides as biomarkers for ALS. These peptides detected in subject with ALS but not healthy humans. "Dea" is Citrullination or deamination. "CAM" is carb ami dom ethyl ati on .

UniProtKB Modificati

Protein Name Modified Sequence

Accessions on position

SYA Alanine-tRNA NVGC[CAM]LQEALQLATSFAQLR[Dea]LGDVK (SEQ

P49588 962

ligase ID NO: 314)

[0155] Table 3 : Citrullinated peptides (from humans) as biomarkers for ALS.

Citrullination or deamination. "CAM" is carbamidomethylation.

Modificat

UniProtKB

Protein Name Modified Sequence ion Accessions

position

Delta(3,5)-Delta(2,4)-dienoyl- VIGNQSLVNELAFTAR[Dea]K (SEQ ID NO:

Q13011 230

CoA isomerase 336)

Cellular retinoic acid-binding

P29762 MR[Dea] SSENFDELLK (SEQ ID NO: 337) 11

protein 1

Q9P2T1 GMP reductase 2 YAGGVAEYR[Dea]ASEGK (SEQ ID NO: 338) 286

[0156] Table 4: Correlated citrullinated LysC peptides based on matching mouse sequence to human sequence. "Dea" is Citrullination or deamination. "CAM" is carb ami dom ethyl ati on .

dimethylaminohydrolase 1 395)

Q9H115 Beta-soluble NSF attachment protein ER[Dea]EAVQLMAEAEK (SEQ ID 8,

[0157] Table 5A: Citrullinated peptides observed in chronic neurodegenerative di These citrullinated peptides are brain specific.

[0158] Table 5B. Citrullinated peptides observed in chronic neurodegenerative disease. These citrullinated peptides are not brain specific.

[0159] Table 6A Citmllinated peptide detected in acquired brain injury in human brains. These citrullinated peptides are brain specific

Peptide UniProtKB Protein Name

Accessions

MDIRG[Dea]AVDAAVPTNIIAAK sp|Q9UI12- Isoform 2 of V-type proton ATPase subunit H (SEQ ID NO: 523) 2|VATH HU OS=Homo sapiens GN=ATP6V1H

MAN

TDD YGR[Dea]DL S S VQTLLTK sp|Q13813- Isoform 3 of Spectrin alpha chain, non-erythrocytic 1 (SEQ ID NO: 524) 3|SPTN1 HU OS=Homo sapiens GN=SPTAN1

MAN

R[Dea]LVPGGGATEIELAK (SEQ sp|P50990- Isoform 3 of T-complex protein 1 subunit theta ID NO: 525) 3|TCPQ HU OS=Homo sapiens GN=CCT8

MAN

VR[Dea]LLADPTGAFGK (SEQ ID sp|P30044- Isoform 4 of Peroxiredoxin-5, OS=Homo sapiens NO: 265) 4|PRDX5 HU GN=PRDX5

MAN

R[Dea] GFGFVTFDDHDPVDK (SEQ sp|P22626- Isoform A2 of Heterogeneous nuclear

ID NO: 526) 2|ROA2 HU ribonucleoproteins A2 B 1 OS=Homo sapiens

MAN GN=HNRNP A2B 1

IATPR[Dea] GAASPAQK (SEQ ID sp|P 10636- Isoform Tau-E of Microtubule-associated protein tau NO: 527) 7|TAU HUM OS=Homo sapiens GN=MAPT

AN

RWAEPVELAQEFR[Dea]K (SEQ sp|075489|ND NADH dehydrogenase [ubiquinone] iron-sulfur ID NO: 528) US3 HUMA protein 3, OS=Homo sapiens GN=NDUFS3 PE=1

N SV=1

GADEAALAR[Dea]AELEK (SEQ sp|P07196|NF Neurofilament light polypeptide OS=Homo sapiens ID NO: 529) L HUMAN GN=NEFL PE=1 SV=3

QIVWNGPVGVFEWEAFAR[Dea] G sp|P00558|PG Phosphoglycerate kinase 1 OS=Homo sapiens TK (SEQ ID NO: 530) Kl HUMAN GN=PGK1 PE=1 SV=3

PMQFLGDEETVR[Dea]K (SEQ ID sp|P18669|PG Phosphoglycerate mutase 1 OS=Homo sapiens NO: 531) AMI HUMA GN=PGAM1 PE=1 SV=2

N

R[Dea] GFGFVYFQSHD AADK sp|Q4VXU2|P Polyadenylate-binding protein 1-like OS=Homo (SEQ ID NO: 532) AP1L HUMA sapiens GN=PABPC1L PE=2 SV=1

N

VPDFSDYRpea]RAEVLDSTK sp|P0C7P4|UC Putative cytochrome b-cl complex subunit Rieske- (SEQ ID NO: 533) RIL HUMAN like protein 1 OS=Homo sapiens GN=UQCRFS1P1

PE=5 SV=1

R[Dea]DVAGDASESALLK (SEQ ID sp|P13637|AT Sodium/potassium-transporting ATPase subunit NO: 534) 1A3 HUMA alpha-3 OS=Homo sapiens GN=ATP1A3 PE=1

N SV=3

DLTSVMR[Dea]LLSK (SEQ ID NO: sp|Q01082|SP Spectrin beta chain, non-erythrocytic 1 OS=Homo 535) TB2 HUMA sapiens GN=SPTBN1 PE=1 SV=2

N

FMELLEPLSER[Dea]K (SEQ ID sp|Q01082|SP Spectrin beta chain, non-erythrocytic 1 OS=Homo NO: 536) TB2 HUMA sapiens GN=SPTBN1 PE=1 SV=2

N

ER[Dea]VEAVNMAEGIIHDTETK sp|P38646|GR Stress-70 protein, OS=Homo sapiens GN=HSPA9 (SEQ ID NO: 537) P75 HUMAN PE=1 SV=2

HVLSGTLGIPEHTYR[Dea] SR[Dea] sp|P04216|TH Thy-1 membrane glycoprotein OS=Homo sapiens VTLSNQPYIK (SEQ ID NO: 538) Yl HUMAN GN=THY1 PE=1 SV=2

MIIYR[Dea]DLISHDELFSDIYK sp|P13693|TC Translationally-controlled tumor protein OS=Homo (SEQ ID NO: 539) TP HUMAN sapiens GN=TPT1 PE=1 SV=1

TGGAVDR[Dea]LTDTSK (SEQ ID sp|Q9BW30|T Tubulin polymerization-promoting protein family NO: 540) PPP3 HUMA member 3 OS=Homo sapiens GN=TPPP3 PE=1

N SV=1

SSEEAVR[Dea]EVHR[Dea]LIEGRA sp|094811|TP Tubulin polymerization-promoting protein

PVISGVTK (SEQ ID NO: 541) PP HUMAN OS=Homo sapiens GN=TPPP PE=1 SV=1

LLIIGTTSR[Dea]K (SEQ ID NO: sp|P46459|NS Vesicle-fusing ATPase OS=Homo sapiens GN=NSF 542) F HUMAN PE=1 SV=3

WGDP VTR[Dea] VLDD GELL VQQT sp|P46459|NS Vesicle-fusing ATPase OS=Homo sapiens GN=NSF Peptide UniProtKB Protein Name

Accessions

K (SEQ ID NO: 543) F_HUMAN PE=1 SV=3

ITR[Dea]VEMLEIIEAIYK (SEQ ID sp|P62760|VIS Visinin-like protein 1 OS=Homo sapiens

NO: 544) LI HUMAN GN=VSNL1 PE=1 SV=2

FPR[dea]YAEIVHLTLPDGT (SEQ sp|P21281|VA V-type proton ATPase subunit B, brain isoform ID NO: 545) TB2 HUMA OS=Homo sapiens GN=ATP6V1B2 PE=1 SV=3

N

[0160] Table 6B. Citrullinated peptide detected in acquired brain injury in human brains. These citrullinated peptides are not brain specific

[0161] Table 7: Secreted citrullinated human proteins in chronic brain disease

[0162] Table 8: Non-secreted citrullinated human proteins in chronic brain disease Q99798 Aconitate hydratase,

014810 Complexin-1;

Q6PUV4 Complexin-2;

Q9Y2J2 Band 4.1-like protein 3;

Q92777 Synapsin-2;

Q2M2I8 AP2 -associated protein kinase 1;

P35611 Alpha-adducin;

P63010 AP-2 complex subunit beta;

015075 Serine/threonine-protein kinase DCLK1;

094925 Glutaminase kidney isoform,

P50213 Isocitrate dehydrogenase [NAD] subunit alpha

Q08209 Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform;

[0163] Table 9: Secreted citrullinated human proteins in acute brain disease

[0164] Table 10: Non-secreted citrullinated human proteins in acute brain disease

References for Example 4

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5. Uysal H, Nandakumar KS, Kessel C, Haag S, Carlsen S, Burkhardt H, et al. Antibodies to citrullinated proteins: molecular interactions and arthritogenicity. Immunol Rev 2010, 233 :9-33. Okada Y, Wu D, Trynka G, Raj T, Terao C, Ikari K, et al. Genetics of rheumatoid arthritis contributes to biology and drug discovery. Nature 2014, 506:376-381.

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Liu Y, Huttenhain R, Collins B, Aebersold R. Mass spectrometric protein maps for biomarker discovery and clinical research. Expert Rev Mol Diagn 2013, 13 :811-825. Escher C, Reiter L, MacLean B, Ossola R, Herzog F, Chilton J, et al. Using iRT, a normalized retention time for more targeted measurement of peptides. Proteomics 2012, 12: 1111-1121.

Crowgey EL, Stabley DL, Chen C, Huang H, Robbins KM, Poison SW, et al. An integrated approach for analyzing clinical genomic variant data from next-generation sequencing. J Biomol Tech 2015, 26: 19-28.

Hensen SM, Boelens WC, Bonger KM, van Cruchten RT, van Delft FL, Pruijn GJ. Phenylglyoxal-based visualization of citrullinated proteins on Western blots. Molecules 2015, 20:6592-6600.

Hensen SM, Pruijn GJ. Methods for the detection of peptidylarginine deiminase (PAD) activity and protein citrullination. Mol Cell Proteomics 2014, 13 :388-396.

Stensland M, Holm A, Kiehne A, Fleckenstein B. Targeted analysis of protein citrullination using chemical modification and tandem mass spectrometry. Rapid Commun Mass Spectrom 2009, 23 :2754-2762. [0165] The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.

[0166] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

[0167] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

[0168] Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context. [0169] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.

[0170] It is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

[0171] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0172] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0173] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention.