ANALYSIS OF UBIQUITINATED POLYPEPTIDES

Abstract

antibody reagents that specifically bind to peptides carrying a ubiquitin remnant from a digested or chemically treated biological sample. The reagents allow the technician to identify ubiquitinated polypeptides as well as the sites of ubiquitination on them. The reagents are preferably employed in proteomic analysis using mass spectrometry. The antibody reagents specifically bind to the remnant of ubiquitin (i.e., a diglycine modified epsilon amine of lysine) left on a peptide which as been generated by digesting or chemically treating ubiquitinated proteins. The inventive antibody reagents' affinity to the ubiquitin remnant does not depend on the remaining amino acid sequences flanking the modified (i.e., ubiquitinated) lysine, i.e., they are context independent.

Description

RELATED APPLICATIONS

[0001] This applications claims benefit from U.S. provisional patent application Ser. No. 61/286,486 filed Dec. 15, 2009, pending, the entire disclosure of which is hereby incorporated by reference. This application is also a continuation-in-part of U.S. Ser. No. 11/823,775 filed Jun. 28, 2007, pending, which itself is a divisional application of U.S. Ser. No. 10/777,893, filed Feb. 12, 2004, now U.S. Pat. No. 7,300,753, which itself is a is a continuation-in-part of U.S. Ser. No. 10/175,486, filed Jun. 19, 2002, now U.S. Pat. No. 7,198,896, which itself claims priority to U.S. Ser. No. 60/299,893, filed Jun. 21, 2001, and U.S. Ser. No. 60/337,012, filed Nov. 8, 2001, both expired, the entire disclosures of each of which are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention provides methods, reagents and kits for analyzing polypeptides and their modifications from biological samples. In particular, the invention provides compositions, kits and methods for detecting ubiquitinated polypeptides and ubiquitination sites in proteins.

BACKGROUND OF THE INVENTION

[0003] Personalized medicine is the application of genomic and molecular data to better target the delivery of health care to specific patients, facilitate the discovery and clinical testing of new products, and help determine a person's predisposition to a particular disease or condition.

[0004] On a technical level, personalized medicine depends on the identification and detection of proteins, genes and genetic variation ("biomarkers") that play a role in a given disease. Rodland, Clin Biochem. 2004 July; 37(7):579-83. The presence or absence of certain biomarkers is then correlated with the incidence of a particular disease or disease predisposition. However, currently available methods for biomarker analysis are associated with long waiting periods, high cost and numerous technical hurdles.

[0005] The current standard for protein detection and/or quantification is based on immunoreactive detection (Western analysis). However, this technique requires the availability of an appropriately specific antibody. In addition, many antibodies only recognize proteins in an unfolded (denatured) form, cross-reactivity can be severely limiting, and quantification is generally relative.

[0006] The development of methods and instrumentation for automated, data-dependent electrospray ionization (ESI) tandem mass spectrometry (MS/MS) in conjunction with microcapillary liquid chromatography (LC) and database searching has significantly increased the sensitivity and speed of the identification of gel-separated proteins. Microcapillary LC-MS/MS has been used successfully for the large-scale identification of individual proteins directly from mixtures without gel electrophoretic separation (Link et al., 1999; Opitek et al., 1997). However, while these approaches accelerate protein identification, quantities of the analyzed proteins cannot be easily determined, and these methods have not been shown to substantially alleviate the dynamic range problem also encountered by the 2DE/MS/MS approach. Therefore, low abundance proteins in complex samples are also difficult to analyze by the microcapillary LC/MS/MS method without their prior enrichment.

[0007] Protein ubiquitination is the one of the most common of all post-translational modifications. Ubiquitin is a highly conserved 76 amino acid protein which is linked to a protein target after a cascade of transfer reactions. Ubiquitin is activated through the formation of a thioester bond between its C-terminal glycine and the active site cysteine of the ubiquitin activating protein, E1 (Hershko, 1991, Trends Biochem. Sci. 16(7): 265-8). In subsequent trans-thiolation reactions, Ubiquitin is transferred to a cysteine residue on a ubiquitin conjugating enzyme, E2 (Hershko, et al., 1983, J. Biol. Chem. 267: 8807-8812). In conjunction with E3, a ubiquitin polypeptide ligase, E2 then transfers ubiquitin to a specific polypeptide target (see, e.g., Scheffner, et al., 1995, Nature 373(6509): 81-3), forming an isopeptide bond between the C-terminal glycine of ubiquitin and the ε-amino group of a lysine present in the target (See FIG. 1).

[0008] The covalent attachment of ubiquitin to cellular polypeptides, in most cases, marks them for degradation by a multi-polypeptide complex called a proteosome. The ubiquitin-proteosome system is the principal mechanism for the turnover of short-lived polypeptides, including regulatory polypeptides (Weissman, 2001, Nat. Rev. Mol. Cell. Biol. 2: 169-78). Some known targets of ubiquitination include: cyclins, cyclin-dependent kinases (CDK's), NFκB, cystic fibrosis transduction receptor, p53, ornithine decarboxylase (ODC), 7-membrane spanning receptors, Cdc25 (phosphotyrosme phosphatase), Rb, Gα, c-Jun and c-Fos. Polypeptides sharing consensus sequences such as PEST sequences, destruction boxes, and F-boxes generally are also targets for ubiquitin-mediated degradation pathways (see, e.g., Rogers, et al., 1986, Science 234: 364-368; Yamano, et al., 1998, The EMBO Journal 17: 5670-5678; Bai, et al., 1996, Cell 86: 263-274).

[0009] Ubiquitin has been implicated in a number of cellular processes including: signal transduction, cell-cycle progression, receptor-mediated endocytosis, transcription, organelle biogenesis, spermatogenesis, response to cell stress, DNA repair, differentiation, programmed cell death, and immune responses (e.g., inflammation). Ubiquitin also has been implicated in the biogenesis of ribosomes, nucleosomes, peroxisomes and myofibrils. Thus, ubiquitin can function both as signal for polypeptide degradation and as a chaperone for promoting the formation of organelles (see, e.g., Fujimuro, et al., 1997, Eur. J. Biochem. 249: 427-433).

[0010] Deregulation of ubiquitination has been implicated in the pathogenesis of many different diseases. For example, abnormal accumulations of ubiquitinated species are found in patients with neurodegenerative diseases such as Alzheimer's as well as in patients with cell proliferative diseases, such as cancer (see, e.g., Hershko and Ciechanover, 1998, Annu Rev. Biochem. 67: 425-79; Layfield, et al., 2001, Neuropathol. Appl. Neurobiol. 27:171-9; Weissman, 1997, Immunology Today 18(4): 189).

[0011] While the importance of its biological role is well appreciated, the ubiquitin pathway is inherently difficult to study. Generally, studies of ubiquitination have focused on particular polypeptides. For example, site-directed mutagenesis has been used to evaluate critical amino acids which form the "destruction boxes", or "D-boxes", of cyclin, sites which are rapidly poly-ubiquitinated when cyclin is triggered for destruction. See, e.g., Yamano, et al., 1998, The EMBO Journal 17: 5670-5678; Amon et al., 1994, Cell 77: 1037-1050; Glotzer, et al., 1991, Nature 349: 132-138; King, et al., 1996, Mol. Biol. Cell 7:1343. Corsi, et al., 1997, J. Biol. Chem. 272(5): 2977-2883, which describe a Western blotting approach to identify ubiquitination sites. In this technique, crude radiolabeled α-spectrin fractions were ubiquitinated in vitro, digested with proteases, and electrophoresed on gels. Ubiquitinated peptides were identified by their differences in mass from peptides generated by digestion of non-ubiquitinated α-spectrin.

[0012] Although mass spectrometry offers a powerful tool for identifying ubiquitin substrates, a number of unresolved issues remain. Despite many advances, MS data is inherently biased toward more abundant substrates. The effects of ubiquitin epitope tags used to enriched ubiqunated proteins remain incompletely understood, including whether purification biases exist and whether ubiquitin pathway enzymes utilize tagged and wild-type ubiquitin with equal efficiency. It is also not clear if ubiquitin-binding proteins or ubiquitin antibodies may work efficiently as affinity reagents in order to lessen the need for epitope. Kirkpatrick et al., Nat Cell Biol. 2005 August; 7(8): 750-757.

SUMMARY OF THE INVENTION

[0013] One aspect of the invention relates to a method for determining the presence of at least one ubiquitinated polypeptide in a biological sample comprising: Contacting the sample with at least one hydrolyzing agent, wherein the hydrolyzing agent is capable of cleaving a ubiquitinated polypeptide to produce at least one ubiquitin remnant peptide, to obtain a hydrolyzed sample; Contacting the hydrolyzed sample with a substrate comprising an at least one immobilized binding partner; wherein the at least one immobilized binding partner preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant; Removing the hydrolyzed sample from the substrate in a manner such that the at least one ubiquitin remnant peptide would remain bound to the immobilized binding partner; Contacting the substrate with an elution solution, wherein the least one ubiquitin remnant peptide would dissociate from the immobilized binding partner into the elution solution; and Determining the presence of a least one ubiquitinated polypeptide in the biological sample when the elution solution contains the at least one least ubiquitin remnant peptide.

[0014] In one embodiment of this aspect of the invention the determining is performed by LC, MS and preferably LC-MS/MS. In a further embodiment, the amino acid sequence of at least one ubiquitin remnant peptide present in the elution solution, is determined. In yet another embodiment, the sequence is compared to the sequence of the ubiquitinated polypeptide and the site of ubiquitination in the ubiquitinated polypeptide is thereby determined. In still a further embodiment, the elution solution further comprises at least one standard peptide, wherein the at least one standard peptide has the substantially the same amino acid sequence as the at least one distinct peptide but a different measured accurate mass.

[0015] Another aspect of the invention relates to an isolated antibody that preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody. In still yet another embodiment, the antibody is selected from the group consisting of single chain Fvs (sdFvs), Fab fragments, Fab' fragments, F(ab')₂, disulfide linked Fvs (sdFvs), Fvs, and fragments thereof. In yet another embodiment, the antibody comprises a polypeptide of SEQ ID NO: 1. In a further embodiment, the antibody comprises a polypeptide of SEQ ID NO: 2. In yet another embodiment, the antibody comprises a light chain polypeptide of SEQ ID NO: 2 and a heavy chain polypeptide of SEQ ID NO: 1. In still another embodiment, the antibody comprises an antigen binding site comprising the variable region of the heavy chain set forth in SEQ ID NO: 1. In still a further embodiment, the antibody comprises an antigen binding site comprising the variable region of the light chain set forth in SEQ ID NO: 2.

[0016] Another aspect of the invention relates to an isolated nucleic acid encoding an antibody that preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant.

[0017] A further aspect of the invention relates to a cell comprising a nucleic acid, preferably in the form of a vector, that encodes an antibody that preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant.

[0018] Another aspect of the invention relates to the isolated ubiquitin remnant peptides listed in Table 4 and fragments and variants thereof.

[0019] Another aspect of the invention relates to nucleic acids encoding the ubiquitin remnant peptides listed in Table 4 and fragments and variants thereof.

[0020] Yet a further aspect of the invention relates to a method for determining whether a patient is has or is likely to have or develop a disease associated with a least one ubiquitinated polypeptide comprising: obtaining a biological sample from the patient; Contacting the sample with at least one hydrolyzing agent, wherein the hydrolyzing agent is capable of cleaving a ubiquitinated polypeptide to produce at least one ubiquitin remnant peptide, to obtain a hydrolyzed sample; Contacting the hydrolyzed sample with a substrate comprising an at least one immobilized binding partner; wherein the at least one immobilized binding partner preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant; Removing the hydrolyzed sample from the substrate in a manner such that the at least one ubiquitin remnant peptide would remain bound to the immobilized binding partner; Contacting the substrate with an elution solution, where in the least one ubiquitin remnant peptide would dissociate from the immobilized binding partner into the elution solution; and Determining the presence of a least one ubiquitinated polypeptide in the biological sample when the elution solution contains the at least one least ubiquitin remnant peptide; Determining that the patient is has or is likely to have or develop the disease associated with a least one ubiquitinated polypeptide if the least one ubiquitinated polypeptide is present in the biological sample.

[0021] Another aspect of the invention relates to a method for determining whether a disease is associated with at least one ubiquitinated polypeptide comprising Obtaining a biological sample from a patient having the disease; Contacting the sample with at least one hydrolyzing agent, wherein the hydrolyzing agent is capable of cleaving a ubiquitinated polypeptide to produce at least one ubiquitin remnant peptide, to obtain a hydrolyzed sample; Contacting the hydrolyzed sample with a substrate comprising an at least one immobilized binding partner; wherein the at least one immobilized binding partner preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant; Removing the hydrolyzed sample from the substrate in a manner such that the at least one ubiquitin remnant peptide would remain bound to the immobilized binding partner; Contacting the substrate with an elution solution, where in the least one ubiquitin remnant peptide would dissociate from the immobilized binding partner into the elution solution; Determining the presence of a least one ubiquitinated polypeptide in the biological sample when the elution solution contains the at least one least ubiquitin remnant peptide; and Determining that the disease is associated with the presence of the at least one ubiquitinated polypeptide if the least one ubiquitinated polypeptide is absent in the biological sample of a healthy individual.

[0022] Still another aspect of the invention relates to a method for determining whether a disease is associated with at least one ubiquitin remnant peptide Obtaining a biological sample from a patient having the disease to obtain a disease biological sample; Obtaining a biological sample from a healthy patient to obtains a healthy biological sample; Contacting the disease biological sample with at least one hydrolyzing agent, wherein the hydrolyzing agent is capable of cleaving a ubiquitinated polypeptide to produce the least one ubiquitin remnant peptide, to obtain a disease hydrolyzed sample; Contacting the healthy biological sample with at least one hydrolyzing agent, wherein the hydrolyzing agent is capable of cleaving a ubiquitinated polypeptide to produce the least one ubiquitin remnant peptide, to obtain a healthy hydrolyzed sample; Contacting the disease hydrolyzed sample with a substrate comprising an at least one immobilized binding partner; wherein the at least one immobilized binding partner preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant; Removing the disease hydrolyzed sample from the substrate in a manner such that the at least one ubiquitin remnant peptide would remain bound to the immobilized binding partner; Contacting the substrate with an elution solution, where in the least one ubiquitin remnant peptide would dissociate from the immobilized binding partner into the elution solution; and Determining the presence of the a least one ubiquitin remnant peptide in the elution solution; Determining that the disease is associated with the presence of the at least one ubiquitin remnant peptide if the least one ubiquitin remnant peptide is absent in the healthy biological sample.

BRIEF DESCRIPTION OF THE FIGURES

[0023] A more complete understanding of the present invention may be obtained by reference to the accompanying drawings, when considered in conjunction with the subsequent detailed description. The embodiments illustrated in the drawings are intended only to exemplify the invention and should not be construed as limiting the invention to the illustrated embodiments, in which:

[0024] FIG. 1 depicts a cartoon of the formation of a ubiquitin remnant

[0025] FIG. 2 shows a heat map illustrating the frequency of amino acids found with the BL4936 polyclonal antibody in a study of four mouse tissues. Altogether 1458 non-redundant peptides were included in this frequency map. The map clearly shows there are no strongly preferred amino acids at least seven residues to the amino-terminal side of K(GG) modification sites (-7 to -1 in the figure) or at least seven residues to the carboxyl-terminal side of K(GG) modification sites.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The inventors have discovered antibody reagents that specifically bind peptides carrying a ubiquitin remnant from a digested or chemically treated biological sample. See also U.S. application Ser. No. 12/455,496 (which is incorporated by reference in its entirety for all purposes and without limitation).

[0027] These reagents allow the technician to identify ubiquitinated polypeptides as well as the sites of ubiquitination on them. The reagents are preferably employed in proteomic analysis using mass spectrometry. The antibody reagents (in both polyclonal and monoclonal form) specifically bind the remnant of ubiquitination, i.e., a diglycine modified epsilon amine of lysine left on a peptide which as been generated by digesting or chemically treating ubiquitinated proteins. The inventive antibody reagents' affinity to the ubiquitin remnant does not depend on the remaining amino acid sequences flanking the modified lysine, i.e., they are "context independent". In addition, the antibodies of the invention do not cross react with peptides lacking the ubiquitin remnant. See for example, U.S. Pat. Nos. 6,441,140; 6,982,318; 7,198,896; 7,259,022; 7,300,753; 7,344,714; U.S. Ser. No. 11/484,485, all herein incorporated by reference in their entirety.

[0028] Notwithstanding the low abundance of ubiquitinated polypeptides in biological samples, the invention allows for high-throughput MS identification of ubiquitination sites. Immunoaffinity purification (IAP) with the inventive antibodies enrich those ubiquitinated peptides derived from the ubiquitinated portion of polypeptides relative to peptides lacking ubiquitination sites, as well as peptides from proteins which strongly interact with ubiquitin or ubiquitinated proteins, thereby significantly reducing the complexity of the peptide mixture. The purified digest sample can be directly applied to tandem MS for efficient peptide sequence analysis and protein identification to reveal ubiquitinated polypeptides and their sites of ubiquitination.

[0029] Prior to describing various embodiments of the current invention, the following definitions are provided:

[0030] As used herein the term "peptide" or "polypeptide" refers to a polymer formed from the linking, in a defined order, of preferably, α-amino acids, D-, L-amino acids, and combinations thereof. The link between one amino acid residue and the next is referred to as an amide bond or a peptide bond. Proteins are polypeptide molecules (or having multiple polypeptide subunits). The distinction is that peptides are preferably short and polypeptides/proteins are preferably longer amino acid chains. The term "protein" is intended to also encompass derivatized molecules such as glycoproteins and lipoproteins as well as lower molecular weight polypeptides.

[0031] As used herein, the term "ubiquitinated polypeptide" refers to a polypeptide bound to ubiquitin, a ubiquitin-like protein (e.g., NEDD8 or ISG15) or a portion thereof. Preferably, ubiquitination is the formation an isopeptide bond between the C-terminal glycine of ubiquitin (or ubiquitin-like protein see e.g., J Proteome Res. 2008 March; 7(3):1274-87) and the ε-amino group of a lysine present in the target. (See e.g., FIG. 1).

[0032] As used herein, a "ubiquitin remnant" or a "ubiquitin tag" is that portion of a ubiquitinated polypeptide which remains attached to the digestion product of the ubiquitinated polypeptide which has been exposed to a hydrolyzing agent such as trypsin. Preferably, the ubiquitin remnant is a diglycine modified epsilon amine of lysine, which adds about 114 daltons to the mass of the lysine residue (see FIG. 1). It is also referred to herein as "K(GG)." Trypsin digestion of neddylated proteins leaves the same K(GG) remnant as trypsin digestion of protein that is attached to ubiquitin.

[0033] A "ubiquitin remnant peptide" is the product that results from the digestion of a ubiquitinated polypeptide with a hydrolyzing agent such as trypsin, i.e., a peptide containing at least one ubiquitin remnant. In the preferred embodiment of the invention, a binding partner is used that specifically recognizes and binds to a ubiquitin remnant peptide but does not cross react with other peptides having the same amino acid sequence but which lack the ubiquitin remnant. The preferred binding partner is an anti-ubiquitin remnant peptide antibody or fragment thereof.

[0034] The invention also encompasses the novel ubiquitin remnant peptides disclosed herein in Table 4 as well as fragments and variants thereof.

[0035] The term "variant" as used herein relative to ubiquitin remnant peptides, refers to a peptide having a ubiquitin remnant that possesses a similar or identical amino acid sequence as a ubiquitin remnant peptide (e.g., one disclosed in Table 4). A variant having a similar amino acid sequence refers to a peptide comprising, or alternatively consisting of, an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the predicate ubiquitin remnant peptide. Peptide variants also include those having a deletion, substitution and/or addition of about 1 to about 2; about 1 to about 3; or about 1 to about 4 amino acids relative to the predicate ubiquitin remnant peptide.

[0036] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity number of identical overlapping positions/total number of positions×100%). In one embodiment, the two sequences are the same length.

[0037] The term "fragment" as used herein refers to a peptide comprising a ubiquitin remnant and an amino acid sequence of at least 3 amino acid residues, at least 5 amino acid residues, at least 7 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues of a ubiquitin remnant peptide.

[0038] The invention also includes nucleic acids that encode for the ubiquitin remnant peptides disclosed herein in Table 4 as well as fragments and variants thereof.

[0039] As used herein, the term "biological sample" refers to a readily obtainable mixture of a plurality of polypeptides present in varying concentrations. Preferred biological samples have about 5,000 to about 20,000 different polypeptides. More preferably, biological samples have about 7,500 to about 15,000 different polypeptides. Most preferably, biological samples have about 10,000 different polypeptides. Generally, such samples are environmental, industrial, veterinary or medical in origin and from an animal, plant, a bacterium, a fungus, a protist or a virus. The preferred biological samples include but are not limited to saliva, mucous, tears, blood, serum, lymph/interstitial fluids, buccal cells, mucosal cells, cerebrospinal fluid, semen, feces, plasma, urine, a suspension of cells, or a suspension of cells and viruses. The most preferred biological samples are mammalian, more preferably human, serum and urine.

[0040] Where the biological sample is blood, serum or lymph/interstitial fluid, the invention envisages an optional step of depleting the biological sample of common and disproportionally over-represented background proteins not suspected of being associated with ubiquitinated polypeptides. Such proteins include but are not limited to albumin, IgG, IgA, transferrin, haptoglobin, and anti-trypsin; or combinations thereof. The skilled artisan will recognized that such a step is carried out by basic affinity chromatography techniques. As used here in the term "depleted" or "depleting" means markedly lessening the concentration of a particular species in a solution, e.g., by more than or about 50%; more than or about 60%; more than or about 65%; more than or about 70%; more than or about 75%; more than or about 80%; more than or about 85%; more than or about 90%; more than or about 92%; more than or about 95%; more than or about 97%; more than or about 98%; more than or about 99%. Alternatively the biological sample may be a subcellular fraction of a cell line or tissue, enriched for specific cellular organelles such as nuclei, cytoplasm, plasma membranes, mitochondria, internal membrane structures, Golgi apparatus, endoplasmic reticulum, etc. or specific tissue organelles such as post-synaptic densities from brain, islets from pancreas, etc.

[0041] As used herein, the term "hydrolyzing agent" refers to any one or combination of a large number of different enzymes, including but not limited to trypsin, Lysine-C endopeptidase (LysC), arginine-C endopeptidase (ArgC), Asp-N, glutamic acid endopeptidase (GluC) and chymotrypsin, V8 protease and the like, as well as chemicals, such as cyanogen bromide. In the subject invention one or a combination of hydrolyzing agents cleave peptide bonds in a protein or polypeptide, in a sequence-specific manner, generating a predictable collection of shorter peptides (a "digest"). A portion of the biological samples are contacted with hydrolyzing agent(s) to form a digest of the biological sample. Given that the amino acid sequences of certain polypeptides and proteins in biological samples are often known and that the hydrolyzing agent(s) cuts in a sequence-specific manner, the shorter peptides in the digest are generally of a predicable amino acid sequence. Preferably, the treatment of a polypeptide with a hydrolyzing agents results in about 2 to about 20, more preferably about 5 to about 15 and most preferably about 10 peptides. If the polypeptide in a biological sample is a ubiquitinated polypeptide, at least one of the resulting peptides in the digest will be a ubiquitin remnant peptide. The preferred hydrolyzing agent is a protease, or chemical which cleaves ubiquitinated proteins in a manner that results in the formation of at least one ubiquitin remnant peptide. Most preferably, the protease is trypsin.

[0042] The term "mass spectrometer" means a device capable of detecting specific molecular species and measuring their accurate masses. The term is meant to include any molecular detector into which a polypeptide or peptide may be eluted for detection and/or characterization. In the preferred MS procedure, a sample, e.g., the elution solution, is loaded onto the MS instrument, and undergoes vaporization. The components of the sample are ionized by one of a variety of methods (e.g., by electrospray ionization or "ESI"), which results in the formation of positively charged particles (ions). The positive ions are then accelerated by a magnetic field. The computation of the mass-to-charge ratio of the particles is based on the details of motion of the ions as they transit through electromagnetic fields, and detection of the ions. The preferred mass measurement error of a mass spectrometer of the invention is 10 ppm or less, more preferable is 7 ppm or less; and most preferably 5 ppm or less.

[0043] Fragment ions in the MS/MS and MS³ spectra are generally highly specific and diagnostic for peptides of interest. In contrast, to prior art methods, the identification of peptide diagnostic signatures provides for a way to perform highly selective analysis of a complex protein mixture, such as a cellular lysate in which there may be greater than about 100, about 1000, about 10,000, or even about 100,000 different kinds of proteins. Thus, while conventional mass spectroscopy would not be able to distinguish between peptides with different sequences but similar m/z ratios (which would tend to co-elute with any labeled standard being analyzed), the use of peptide fragmentation methods and multistage mass spectrometry in conjunction with LC methods, provide a way to detect and quantify target proteins which are only a small fraction of a complex mixture (e.g., present in less than 2000 copies per cell or less than about 0.001% of total cellular protein) through these diagnostic signatures.

[0044] Test peptides are preferably examined by monitoring of a selected reaction in the mass spectrometer. This involves using the prior knowledge gained by the characterization of a standard peptide and then requiring the mass spectrometer to continuously monitor a specific ion in the MS/MS or MS' spectrum for both the peptide of interest and the standard peptide. After elution, the areas-under-the-curve (AUC) for both the standard peptide and target peptide peaks may be calculated. The ratio of the two areas provides the absolute quantification that may then be normalized for the number of cells used in the analysis and the protein's molecular weight, to provide the precise number of copies of the protein per cell.

[0045] As used herein the term, "accurate mass" refers to an experimentally or theoretically determined mass of an ion that is used to determine an elemental formula. For ions containing combinations of the elements C, H, N, O, P, S, and the halogens, with mass less than 200 Unified Atomic Mass Units, a measurement about 5 ppm uncertainty is sufficient to uniquely determine the elemental composition.

[0046] As used herein the term, "predetermined peptide accurate mass" refers to the experimentally determined or calculated accurate mass of a peptide with a known amino acid sequence (along with any associated post-translational modifications). The accurate mass of any such specific amino acid sequence may be readily calculated by one of skill in the art.

[0047] As used herein, "a peptide fragmentation signature" refers to the distribution of mass-to-charge ratios of fragmented peptide ions obtained from fragmenting a peptide, for example, by collision induced disassociation, ECD, LID, PSD, IRNPD, SID, and other fragmentation methods. A peptide fragmentation signature which is "diagnostic" or a "diagnostic signature" of a target protein or target polypeptide is one which is reproducibly observed when a peptide digestion product of a target protein/polypeptide identical in sequence to the peptide portion of a standard peptide, is fragmented and which differs only from the fragmentation pattern of the standard peptide by the mass of the mass-altering label and/or the presence of a ubiquitin remnant. Preferably, a diagnostic signature is unique to the target protein (i.e., the specificity of the assay is at least about 95%, at least about 99%, and preferably, approaches 100%).

[0048] The term "substrate" includes any solid support or phase upon which a binding partner may be immobilized. Preferred supports are those well known in the art of affinity chromatography for example but not limited to polymeric and optionally magnetic beads, polystyrene, sepharose or agarose gel matrices, or nitrocellulose membranes.

[0049] The term "binding partner" refers to any of a large number of different molecules or aggregates. Preferably, a binding partner functions by binding to a polypeptide or peptide in order to enrich it prior to analysis, e.g., by MS, LC-MS, or LC-MS/MS. Preferably, binding partners bind ubiquitin remnant peptides to enrich in a digest. Proteins, polypeptides, peptides, nucleic acids (oligonucleotides and polynucleotides), antibodies, ligands, polysaccharides, microorganisms, receptors, antibiotics, and test compounds (particularly those produced by combinatorial chemistry) may each be a binding partner.

[0050] In the preferred one embodiment, the binding partner is immobilized by being directly or indirectly, covalently or non-covalently bound to the substrate. In another embodiment, the binding partner does not require a substrate and can be used to immuno-precipitate the ubiquitin remnant peptides for example. In a further embodiment, the binding partner can be used to bind ubiquitin remnant peptides in solution. The technician could then enrich for ubiquitin remnant peptides by filtering ubiquitin remnant peptide-binding partner complexes, through size cut-off or size exclusion chromatography for example.

[0051] The preferred binding partner is a "ubiquitin remnant peptide specific antibody" or an "anti-ubiquitin remnant antibody" which specifically yet reversibly binds ubiquitin remnant peptides and does not bind (i.e., cross react with) peptides having the same amino acid sequence but which lack the ubiquitin remnant. As such, the preferred ubiquitin remnant peptide-specific antibodies bind ubiquitin remnant peptides in a context independent manner.

[0052] Accordingly, the invention provides an isolated antibody or binding partner that preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacks the ubiquitin remnant. In some embodiments, the isolated antibody or binding partner specifically binds a ubiquitin remnant peptide but does not specifically bind a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacks the ubiquitin remnant. As used herein, by "specifically binds" is meant that a binding partner or an antibody of the invention interacts with its target molecule (e.g., a ubiquitin remnant peptide), where the interaction is dependent upon the presence of a particular structure (e.g., the antigenic determinant or epitope on the peptide); in other words, the reagent is recognizing and binding to a specific polypeptide structure rather than to all polypeptides in general. In some embodiments, the isolated antibodies or isolated binding partners do not specifically bind to a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacks the ubiquitin remnant.

[0053] The isolated antibodies and/or isolated binding partners of the invention can be used in the methods of the invention.

[0054] It should be understood that the substrate can have a number many different binding partners having a different binding specificity for a different polypeptide, peptide, ubiquitin remnant peptide or epitopes thereof. As such, binding partners might be derived from monoclonal sources or polyclonal sera. Preferably, the substrate has about 2 to about 500, more preferably about 5 to about 400, even more preferably about 10 to about 300 and most preferably about 15 to about 200, yet even more preferably about 20 to about 100, about 25 to about 75 and about 30 to about 60 different binding partners each specifically binding to a different and/or distinct peptide. This allows the technician to simultaneously process and analyze the biological sample for the presence of a large number of polypeptides in a manner not feasible with multiplex PCR or ELISA techniques. Additional methods and reagents for immunoaffinity purification and/or enrichment of peptides containing certain motifs such as the ubiquitin remnant may be found in e.g., in U.S. Pat. Nos. 7,198,896 and 7,300,753.

[0055] The term "antibody" as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds to an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody multimers and antibody fragments, as well as variants (including derivatives) of antibodies, antibody multimers and antibody fragments. The preferred antibody disclosed herein is referred to as D4A7A10.

[0056] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kilodalton) and one "heavy" chain (about 50-70 kilodalton).

[0057] The amino-terminal portion of each chain includes a variable region of about, 80, 85, 90, 95, 100, 105, preferably 100 to 110 or more amino acids primarily responsible for antigen recognition. Herein the terms "heavy chain" and "light chain" refer to the heavy and light chains of an antibody unless otherwise specified. The amino acid sequence of the D4A7A10 heavy chain is set forth in SEQ ID NO: 1. The amino acid sequence of the D4A7A10 light chain is set forth in SEQ ID NO: 2.

[0058] The carboxy-terminal portion of each chain preferably defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light ("VL")/heavy chain ("VH") pair preferably form the antibody binding site. Thus, an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same. The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the heavy and the light chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989).

[0059] A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al. J Immunol. 148:1547 1553 (1992). In addition, bispecific antibodies may be formed as "diabodies" (Holliger et al. "`Diabodies`: small bivalent and bispecific antibody fragments" PNAS USA 90:6444-6448 (1993)) or "Janusins" (Traunecker et al. "Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells" EMBO J 10:3655-3659 (1991) and Traunecker et al. "Janusin: new molecular design for bispecific reagents" Int J Cancer Suppl 7:51-52 (1992)). Production of bispecific antibodies can be a relatively labor intensive process compared with production of conventional antibodies and yields and degree of purity are generally lower for bispecific antibodies.

[0060] Examples of molecules which are described by the term "antibody" herein include, but are not limited to: single chain Fvs (sdFvs), Fab fragments, Fab' fragments, F(ab')₂, disulfide linked Fvs (sdFvs), Fvs, and fragments thereof comprising or alternatively consisting of, either a VL or a VH domain. The term "single chain Fv" or "scFv" as used herein refers to a polypeptide comprising a VL domain of antibody linked to a VH domain of an antibody.

[0061] Antibodies of the invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass of immunoglobulin molecule. Preferably, an antibody of the invention comprises, or alternatively consists of, a VH domain, VH CDR, VL domain, or VL CDR having an amino acid sequence of any one of the antibodies listed in Table 1, or a fragment or variant thereof. In a preferred embodiment, the immunoglobulin is an IgG1 isotype. In another preferred embodiment, the immunoglobulin is an IgG4 isotype. Immunoglobulins may have both a heavy and light chain. An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa or lambda forms. Antibodies of the invention may also include multimeric forms of antibodies. For example, antibodies of the invention may take the form of antibody dimers, trimers, or higher-order multimers of monomeric immunoglobulin molecules. Dimers of whole immunoglobulin molecules or of F(ab')2 fragments are tetravalent, whereas dimers of Fab fragments or scFv molecules are bivalent. Individual monomers withon an antibody multimer may be identical or different, i.e., they may be heteromeric or homomeric antibody multimers. For example, individual antibodies within a multimer may have the same or different binding specificities.

[0062] Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers, and other higher-order antibody multimers. Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to, SMCC [succinimidyl 4-(maleimidomethyl)cyclohexane-1 carboxylate] and SATA [N-succinimidyl S-acethylthio-acetate] (available, for example, from Pierce Biotechnology, Inc. (Rockford, Ill.)) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is given in Ghetie et al., Proceedings of the National Academy of Sciences USA (1997) 94:7509-7514, which is hereby incorporated by reference in its entirety. Antibody homodimers can be converted to Fab'2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao and Kohler, The Journal of Immunology (2002) 25:396-404, which is hereby incorporated by reference in its entirety.

[0063] Alternatively, antibodies can be made to multimerize through recombinant DNA techniques. IgM and IgA naturally form antibody multimers through the interaction with the mature J chain polypeptide. Non-IgA or non-IgM molecules, such as IgG molecules, can be engineered to contain the J chain interaction domain of IgA or IgM, thereby conferring the ability to form higher order multimers on the non-IgA or non-IgM molecules. (see, for example, Chintalacharuvu et al., (2001) Clinical Immunology 101:21-31. and Frigerio et al., (2000) Plant Physiology 123:1483-94, both of which are hereby incorporated by reference in their entireties.) IgA dimers are naturally secreted into the lumen of mucosa-lined organs. This secretion is mediated through interaction of the J chain with the polymeric IgA receptor (pIgR) on epithelial cells. If secretion of an IgA form of an antibody (or of an antibody engineered to contain a J chain interaction domain) is not desired, it can be greatly reduced by expressing the antibody molecule in association with a mutant J chain that does not interact well with pIgR (Johansen et al., The Journal of Immunology (2001) 167:5185-5192 which is hereby incorporated by reference in its entirety). ScFv dimers can also be formed through recombinant techniques known in the art; an example of the construction of scFv dimers is given in Goel et al., (2000) Cancer Research 60:6964-6971 which is hereby incorporated by reference in its entirety. Antibody multimers may be purified using any suitable method known in the art, including, but not limited to, size exclusion chromatography.

[0064] Monoclonal and polyclonal context-independent ubiquitin remnant peptide antibodies have been identified. For example, the invention encompasses the monoclonal and polyclonal antibodies listed in Table 1 and the cell lines engineered to express them or capable of expressing them.

[0065] Further, the present invention encompasses the polynucleotides encoding the anti-ubiquitin remnant peptide antibodies or portions thereof. Molecules encoding e.g., VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of the corresponding region of the inventive antibodies expressed by a cell that specifically bind to ubiquitin remnant peptides but not peptides having the same amino acid sequence but lacking the ubiquitin remnant, or fragments or variants thereof are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies and/or molecules. In specific embodiments, the present invention encompasses antibodies, or fragments or variants thereof that bind to an epitope that comprises the ubiquitin remnant.

[0066] Methods for identifying the complementarity determining regions (CDRs) of an antibody by analyzing the amino acid sequence of the antibody are well known (see, e.g., Wu, T. T. and Kabat, E. A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(1-3):9-16 (October 1997); Morea et al., J Mol Biol. 275(2):269-94 (January 1998); Chothia et al., Nature 342(6252):877-83 (December 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007).

[0067] As one non-limiting example, the following method can be used to identify the CDRs of an antibody.

[0068] For the CDR-L1, the CDR-L1 is approximately 10-17 amino acid residues in length. Generally, the start is at approximately residue 24 (the residue before the 24^th residue is typically a cysteine. The CDR-L1 ends on the residue before a tryptophan residue. Typically, the sequence containing the tryptophan is either Trp-Tyr-Gln, Trp-Leu-Gln Trp-Phe-Gln, or Trp-Tyr-Leu, where the last residue within the CDR-L1 domain is the residue before the TRP in all of these sequences.

[0069] For the CDR-L2, the CDR-L2 is typically seven residues in length. Generally, the start of the CDR-L2 is approximately sixteen residues after the end of CDR-L1 and typically begins on the on the residue after the sequences of Ile-Tyr, Val-Tyr, Ile-Lys, or Ile-Phe.

[0070] For the CDR-L3, the CDR-L3 is typically 7-11 amino acid residues in length. Generally, the domain starts approximately 33 residues after the end of the CDR-L2 domain. The residue before the start of the domain is often a cysteine and the domain ends on the residue before Phe in the sequence Phe-Gly-XXX-Gly (where XXX is the three letter code of any single amino acid).

[0071] For the CDR-H1, the CDR-H1 domain is typically 10-12 amino acid residues in length and often starts on approximately residue 26. The domain typically starts four or five residues after a cysteine residue, and typically ends on the residue before a Trp (the Trp is often found in one of the following sequences: Trp-Val, Trp-Ile, or Trp-Ala.

[0072] For the CDR-H2, the CDR-H2 domain is typically 16 to 19 residues in length and typically starts 15 residues after the final residue of the CDR-H1 domain. The domain typically ends on the amino acid residue before the sequence Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala (which includes, for example, the sequences Lys-Leu-Thr and Arg-Ala-Ala).

[0073] For the CDR-H3, the CDR-H3 domain is typically 3-25 amino acids in length and typically starts 33 amino acid residues after the final residues of the CDR-H2 domain (which is frequently two amino acid residues after a cysteine residue, e.g., a cysteine in the sequence Cys-Ala-Arg). The domain ends on the amino acid immediately before the Trp in the sequence Trp-Gly-XXX-Gly (where XXX is the three letter code of any single amino acid).

[0074] The inventive anti-ubiquitin remnant peptide antibodies may be coupled to a detectable label such as an enzyme, a fluorescent label, a luminescent label, or a bioluminescent label. The present invention also provides anti-ubiquitin remnant peptide antibodies that are coupled to a therapeutic or cytotoxic agent. The present invention also provides anti-PA antibodies which are coupled, directly or indirectly, to a radioactive material.

[0075] In further embodiments, the anti-ubiquitin remnant peptide antibodies of the invention have a dissociation constant (K_D) of 10^-7 M or less for a ubiquitin remnant peptide. In preferred embodiments, the anti-ubiquitin remnant peptide antibodies of the invention have a dissociation constant (K_D) of 10^-9 M or less for a ubiquitin remnant peptide.

[0076] In further embodiments, antibodies of the invention have an off rate (k_off) of 10^-3/sec or less. In preferred embodiments, antibodies of the invention have an off rate (k_off) of 10^-4/sec or less. In other preferred embodiments, antibodies of the invention have an off rate (k_off) of 10^-5/sec or less.

[0077] The present invention also provides panels of the anti-ubiquitin remnant peptide antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) wherein the panel members correspond to one, two, three, four, five, ten, fifteen, twenty, or more different the anti-ubiquitin remnant peptide antibodies of the invention (e.g., whole antibodies, Fabs, F(ab')₂ fragments, Fd fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and scFvs). The present invention further provides mixtures of the anti-ubiquitin remnant peptide antibodies wherein the mixture corresponds to one, two, three, four, five, ten, fifteen, twenty, or more different the anti-ubiquitin remnant peptide antibodies of the invention (e.g., whole antibodies, Fabs, F(ab')₂ fragments, Fd fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and scFvs)). The present invention also provides for compositions comprising, or alternatively consisting of, one, two, three, four, five, ten, fifteen, twenty, or more the anti-ubiquitin remnant peptide antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof). A composition of the invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty, or more amino acid sequences of one or more of the anti-ubiquitin remnant peptide antibodies or fragments or variants thereof. Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one or more antibodies of the invention.

[0078] The present invention also provides for fusion proteins comprising an anti-ubiquitin remnant peptide antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) of the invention, and a heterologous polypeptide (i.e., a polypeptide unrelated to an antibody or antibody domain). Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention. A composition of the present invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention.

[0079] Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention.

[0080] The term "elution solution" refers to a solution that when brought into contact with the binding partner, results in the dissociation of the polypeptide or peptide and preferably the ubiquitin remnant peptide from the binding partner into the elution solution. Determining the salt, pH and ionic conditions necessary for such functionality is well with the ordinary skill in the art. Preferably, the elution solution is enriched for polypeptides and peptides which were bound to the binding partners relative to the polypeptides and peptides of the digest. Preferably, the elution solution has about 500 to about 5000, more preferably about 1000 to about 2000 different peptides. Most preferably, the elution solution is enriched for ubiquitin remnant peptides. Preferably, a portion of the elution solution is directly transferred to a mass spectrometer, LC-MS or LC-MS/MS. Alternatively, the elution solution is subject to further manipulation e.g., to concentrate the peptides and/or polypeptides contained therein. Mechanisms for directing solutions from liquid chromatography to mass spectrometers may be found for example in U.S. Pub. No. 20080217254.

[0081] The term "vaporizing a portion of the elution solution" means that a portion of the elution solution is preferably transferred to a mass spectrometer for vaporization and ionization.

[0082] The term "ionizing" refers to atmospheric pressure chemical ionization (APCI), chemical ionization (CI), electron impact (EI), electrospray ionization (ESI), fast atom bombardment (FAB), field desorption/field ionization (FD/FI), matrix assisted laser desorption ionization (MALDI), and thermospray ionization. The preferred method of ionization is ESI as tends to minimize the propensity of macromolecules to fragment when ionized.

[0083] Preferably in ESI, liquid containing the peptides of interest is dispersed by electrospray into a fine aerosol. Preferred solvents for electrospray ionization are prepared by mixing water with volatile organic compounds (e.g. methanol, acetonitrile). To decrease the initial droplet size, compounds that increase the conductivity (e.g. acetic acid) are preferably added to the solution. Large-flow electrosprays may provide additional nebulization by an inert gas such as nitrogen. The aerosol is sampled into the first vacuum stage of a mass spectrometer through a capillary, which can be heated to aid further solvent evaporation from the charged droplets. Preferably, the solvent evaporates from a charged droplet until it becomes unstable upon reaching its Rayleigh limit. At this point, the droplet preferably deforms and emits charged jets in a process known as Rayleigh fission. During the fission, the droplet loses a small percentage of its mass along with a relatively large percentage of its charge

[0084] As used herein, "ionized molecule" refers to molecules in the elution solution that have become charged and are ready to move into the electric fields that will direct them into the mass analyzer of a mass spectrometer. Preferably, the ionized molecules include ionized polypeptides, peptides and/or ubiquitin remnant peptides present in the elution solution. Most preferably, the ionized molecules are ubiquitin remnant peptides.

[0085] The term "standard peptide" as used herein, refers to a peptide that is 1) recognized as equivalent to a peptide of interest in the digest generated by a hydrolyzing agent, e.g., the ubiquitin remnant peptide, by the appropriate binding partner; and 2) differs from the peptide of interest in a manner that can be distinguished by a mass spectrometer, e.g., by way of a mass-altering label. Preferably, the standard peptide has the same amino acid sequence as the ubiquitin remnant peptide but is synthesized utilizing elemental isotopes. Preferably, those isotopes are ¹⁵N, ¹³C, ¹⁸O or ²H. Alternatively, a standard peptide can 1) have the same amino acid sequence as a ubiquitin remnant peptide yet lack the ubiquitin remnant; and 2) differ from the ubiquitin remnant peptide in a manner that can be distinguished by a mass spectrometer, e.g., by lacking the ubiquitin remnant. Exemplary standard peptides are described in U.S. Pub. No. 20060154318 and 20060148093. One or more standard peptides may be added to the biological sample before or after treatment with a hydrolyzing agent such that it co-elutes with the peptide of interest into the elution solution. The standard peptide can be added directly to the elution solution.

[0086] One aspect of the invention relates to providing methods for determining a site of ubiquitination in a polypeptide. The method comprises obtaining a plurality of ubiquitinated polypeptides; digesting the ubiquitinated polypeptides with a protease, thereby generating a plurality of test peptides; enriching the plurality of test peptides for ubiquitin remnant peptides; and determining the presence of a ubiquitin remnant peptide by mass spectrometry, wherein the presence of the ubiquitin remnant peptide allows the technician to determine a site of ubiquitination of the polypeptide. The test peptide being evaluated can be ionized and/or fragmented prior to the determining step. Preferably, ionizing is performed by electrospray.

[0087] In one embodiment of this aspect of the invention, the method for determining a site of ubiquitination comprises obtaining a plurality of ubiquitinated polypeptides; digesting the ubiquitinated polypeptides with a protease; thereby generating a plurality of test peptides; at least some of which comprise a ubiquitin remnant, enriching the plurality of test peptides for ubiquitin remnant peptides; and identifying a mass difference between a test peptide and a standard peptide comprising a known identical amino acid sequence as the test peptide; the mass difference corresponding to the mass of the ubiquitin remnant, wherein detection of the mass difference indicates a site of ubiquitination in the test peptide.

[0088] In another aspect, the methods further comprise the step of mapping a sequence of a test peptide comprising a ubiquitin remnant to a polypeptide sequence comprising the same amino acid sequence as the test peptide, thereby determining the site of ubiquitination in the polypeptide sequence. In another embodiment, the ubiquitin remnant comprises Gly-Gly amino acid residues and has a mass of about 114 daltons. The methods can be used to detect one or more sites of ubiquitination in a polypeptide, as well as the amount of ubiquitination at particular sites in a population of polypeptides.

[0089] In a further aspect of the invention, ubiquitination sites are identified for a plurality of polypeptides in a first cell and in a second cell and the sites identified in the first cell are compared to those in the second cell. In one aspect, the first cell is a normal cell (e.g., from a healthy patient), while the second cell is from a patient with a pathological condition (e.g., a neurodegenerative disease, cancer, a disease of the immune system). Preferably, the second cell is the target of the pathology (e.g., a tumor cell from a cancer patient; a neural cell from a patient with a neurodegenerative disease). In another embodiment of this aspect of the invention, the second cell differs from the first cell in expressing one or more recombinant DNA molecules, but is otherwise genetically identical to the first cell. In a further embodiment, the site of ubiquitination is correlated with disease and detection of ubiquitination at the site is associated with risk of the disease. In another embodiment, the disease is a neurodegenerative disease, such as Alzheimer's or Pick's disease. In another aspect, the disease is cancer. In a further aspect, the disease is an abnormal immune response or inflammatory disease.

[0090] In another aspect of the invention, the methods disclosed herein are used to identify regulators of ubiquitination pathways. In one embodiment, the methods further comprise contacting a first cell with a compound and comparing ubiquitination sites identified in the first cell with ubiquitination sites in a second cell not contacted with the compound. The compound may be a therapeutic agent for treating a disease associated with an improper state of ubiquitination (e.g., abnormal sites or amounts of ubiquitination). Suitable agents include, but are not limited to, drugs, polypeptides, peptides, antibodies, nucleic acids (genes, cDNA's, RNA's, antisense molecules, siRNA/miRNA constructs, ribozymes, aptamers and the like), toxins, and combinations thereof.

[0091] Preferably, the methods further comprise generating a database comprising data files storing information relating to ubiquitination sites for a plurality of polypeptides for a plurality of different cells. Preferably, the data files also include information relating to amount of ubiquitination of a polypeptide in at least one cell. Additionally, the database comprises data relating to the source of the cell (e.g., such as a patient).

[0092] The invention further provides a computer memory comprising data files storing information relating to ubiquitination sites for a plurality of polypeptides for a plurality of different cells.

[0093] In another aspect of the invention, substantially purified test peptides, preferably ubiquitin remnant peptides, obtained after one or more separation steps are analyzed by a peptide analyzer that evaluates the mass of the peptide or a fragment thereof. Suitable peptide analyzers include, but are not limited to, a mass spectrometer, mass spectrograph, single-focusing mass spectrometer, static field mass spectrometer, dynamic field mass spectrometer, electrostatic analyzer, magnetic analyzer, quadropole analyzer, time of flight analyzer (e.g., a MALDI Quadropole time-of-flight mass spectrometer), Wien analyzer, mass resonant analyzer, double-focusing analyzer, ion cyclotron resonance analyzer, ion trap analyzer, tandem mass spectrometer, liquid secondary ionization MS, and combinations thereof in any order (e.g., as in a multi-analyzer system). Such analyzers are known in the art and are described in, for example, Mass Spectrometry for the Biological Sciences, Burlingame and Carr eds., Human Press, Totowa, N.J.)

[0094] In general, any analyzer can be used that can separate matter according to its anatomic and molecular mass. Preferably, the peptide analyzer is a tandem MS system (an MS/MS system) since the speed of an MS/MS system enables rapid analysis of low femtomole levels of peptide and can be used to maximize throughput.

[0095] In a preferred embodiment of this aspect of the invention, the peptide analyzer comprises an ionizing source for generating ions of a test peptide and a detector for detecting the ions generated. The peptide analyzer further comprises a data system for analyzing mass data relating to the ions generated and for deriving mass data relating to the test peptide.

[0096] A sample comprising a test peptide can be delivered to the peptide analyzer using a delivery mechanism as described above. Interfaces between a sample source (e.g., an HPLC column) and ion source can be direct or indirect. For example, there may be an interface that provides for continuous introduction of the sample to the ion source. Alternatively, sample can be intermittently introduced to the ion source (e.g., in response to feedback from the system processor during the separation process, or while the separation system is off-line).

[0097] In another embodiment, the ion source is an electrospray which is used to provide droplets to the peptide analyzer, each droplet comprising a substantially purified test peptide obtained from previous separation step(s) (e.g., such as HPLC or reversed phase liquid chromatography). During electrospray, a high voltage is applied to a liquid stream causing large droplets to be subdivided into smaller and smaller droplets until a peptide enters the gas phase as an ion. Ionization generally is accomplished when the test peptide loses or gains a proton at one or more sites on the peptide (e.g., at the amino terminus, and/or at lysine and arginine residues). Ionization in electrospray is constant; MALDI can be used to achieve pulsed ionization. Other methods of ionization, include but are not limited to, plasma desorption ionization, thermospray ionization, and fast atom bombardment ionization as are known in the art.

[0098] When MALDI is used, peptides can be delivered to a solid support, e.g., sample plate inserted into the mass spectrometer. The support may comprise a light-absorbent matrix. In another embodiment, a substantially purified ubiquitinated polypeptide is provided on a sample plate and protease digestion occurs on the sample plate prior to ionization. For example, substantially purified ubiquitinated peptides also can be obtained from protease digests as described above and separated by a liquid chromatography method. Preferably, the peptide analyzer further comprises an ion transfer section through which ions are delivered from the ion source to the detector. The ion transfer section comprises an electric and/or magnetic field generator (e.g., an electrode ring) that modulates the acceleration of ions generated by the ionizing source. The electric/magnetic field generator directs ions through the ion transfer section of the peptide analyzer to the ion detector.

[0099] Preferably, the peptide analyzer further comprises an ion trap positioned between the ion transfer section of the analyzer and the detector, for performing one or more operations such as ion storage, ion selection and ion collision. The ion trap can be used to fragment ions produced by the ion source (e.g., causing ions to undergo collisional activated dissociation in the presence of a neutral gas ions, such as helium ions). The ion trap also can be used to store ions in stable orbits and to sequentially eject ions based on their mass-to-charge values (m/z) to the detector. An additional separation section can be provided between the ion trap and detector to separate fragments generated in the ion trap (e.g., as in tandem MS). The detector detects the signal strength of each ion (e.g., intensity), which is a reflection of the amount of protonation of the ion.

[0100] The peptide analyzer additionally preferably is associated with data system for recording and processing information collected by the detector. The data system can respond to instructions from a processor in communication with the separation system and also can provide data to the processor. Preferably, the data system includes one or more of: a computer; an analog to digital conversion module; and control devices for data acquisition, recording, storage and manipulation. More preferably, the device further comprises a mechanism for data reduction, i.e., a device to transform the initial digital or analog representation of output from the analyzer into a form that is suitable for interpretation, such as a graphical display, a table of masses, a report of abundances of ions, etc.)

[0101] The data system can perform various operations such as signal conditioning (e.g., providing instructions to the peptide analyzer to vary voltage, current, and other operating parameters of the peptide analyzer), signal processing, and the like. Data acquisition can be obtained in real time, e.g., at the same time mass data is being generated. However, data acquisition also can be performed after an experiment, e.g., when the mass spectrometer is off line.

[0102] The data system can be used to derive a spectrum graph in which relative intensity (i.e., reflecting the amount of protonation of the ion) is plotted against the mass to charge ratio (m/z ratio) of the ion or ion fragment. An average of peaks in a spectrum can be used to obtain the mass of the ion (e.g., peptide) (see, e.g., McLafferty and Turecek, 1993, Interpretation of Mass Spectra, University Science Books, CA).

[0103] Mass spectra can be searched against a database of reference peptides of known mass and sequence to identify a reference peptide which matches a test peptide (e.g., comprises a mass which is smaller by the amount of mass attributable to a ubiquitin remnant). The database of standard peptides can be generated experimentally, e.g., digesting non-ubiquitinated peptides and analyzing these in the peptide analyzer. The database also can be generated after a virtual digestion process, in which the predicted mass of peptides is generated using a suite of programs such as PROWL (e.g., available from ProteoMetrics, LLC, New York; N.Y.). A number of database search programs exist which can be used to correlate mass spectra of test peptides with amino acid sequences from polypeptide and nucleotide databases, including, but not limited to: the SEQUEST program (Eng, et al., J. Am. Soc. Mass Spectrom. 5: 976-89; U.S. Pat. No. 5,538,897; Yates, Jr., III, et al., 1996, J. Anal. Chem. 68(17): 534-540A), available from Finnegan Corp., San Jose, Calif.

[0104] Data obtained from fragmented peptides can be mapped to a larger peptide or polypeptide sequence by comparing overlapping fragments. Preferably, a ubiquitinated peptide is mapped to the larger polypeptide from which it is derived to identify the ubiquitination site on the polypeptide. Sequence data relating to the larger polypeptide can be obtained from databases known in the art, such as the nonredundant protein database compiled at the Frederick Biomedical Supercomputing Center at Frederick, Md.

[0105] In another aspect of the invention, the amount and location of ubiquitination is compared to the presence, absence and/or quantity of other types of polypeptide modifications. For example, the presence, absence, and/or quantity of: phosphorylation, sulfation, glycosylation, and/or acetylation can be determined using methods routine in the art (see, e.g., Rossomando, et al., 1992, Proc. Natl. Acad. Sci. USA 89: 5779-578; Knight et al., 1993, Biochemistry 32: 2031-2035; U.S. Pat. No. 6,271,037). The amount and locations of one or more modifications can be correlated with the amount and locations of ubiquitination sites. Preferably, such a determination is made for multiple cell states.

[0106] Knowledge of ubiquitination sites can be used to identify compounds that modulate particular ubiquitinated polypeptides (either preventing or enhancing ubiquitination, as appropriate, to normalize the ubiquitination state of the polypeptide). Thus, in one aspect, the method described above may further comprise contacting a first cell with a compound and comparing ubiquitination sites/amounts identified in the first cell with ubiquitination sites/amounts in a second cell not contacted with the compound. Suitable cells that may be tested include, but are not limited to: neurons, cancer cells, immune cells (e.g., T cells), stem cells (embryonic and adult), undifferentiated cells, pluripotent cells, and the like. In one preferred aspect, patterns of ubiquitination are observed in cultured cells, such as P19 cells, pluripotent embryonic carcinoma cells capable of differentiating into cardiac cells and skeletal myocytes upon exposure to DMSO (see Montross, et al., J. Cell Sci. 113 (Pt. 10): 1759-70).

[0107] Compounds which can be evaluated include, but are not limited to: drugs; toxins; proteins; polypeptides; peptides; amino acids; antigens; cells, cell nuclei, organelles, portions of cell membranes; viruses; receptors; modulators of receptors (e.g., agonists, antagonists, and the like); enzymes; enzyme modulators (e.g., such as inhibitors, cofactors, and the like); enzyme substrates; hormones; nucleic acids (e.g., such as oligonucleotides; polynucleotides; genes, cDNAs; RNA; antisense molecules, ribozymes, aptamers); and combinations thereof. Compounds also can be obtained from synthetic libraries from drug companies and other commercially available sources known in the art (e.g., including, but not limited to the LeadQuest® library) or can be generated through combinatorial synthesis using methods well known in the art. A compound is identified as a modulating agent if it alters the site of ubiquitination of a polypeptide and/or if it alters the amount of ubiquitination by an amount that is significantly different from the amount observed in a control cell (e.g., not treated with compound).

[0108] In further aspect of the invention, the ubiquitination states (e.g., sites and amount of ubiquitination) of first and second cells are evaluated. Preferably, the second cell differs from the first cell in expressing one or more recombinant DNA molecules, but is otherwise genetically identical to the first cell. Alternatively, or additionally, the second cell can comprise mutations or variant allelic forms of one or more genes. In one aspect, DNA molecules encoding regulators of the ubiquitin pathway can be introduced into the second cell (e.g., E1, E2, E3, deubiquitinating proteins, fragments thereof, mutant forms thereof, variants, and modified forms thereof, or compounds identified as above) and alterations in the ubiquitination state in the second cell can be determined. DNA molecules can be introduced into the cell using methods routine in the art, including, but not limited to: transfection, transformation, electroporation, electrofusion, microinjection, and germline transfer.

[0109] The invention also provides methods for generating a database comprising data files for storing information relating to diagnostic peptide fragmentation signatures. Preferably, data in the data files include one or more peptide fragmentation signatures characteristic or diagnostic of a cell state (e.g., such as a state which is characteristic of a disease, a normal physiological response, a developmental process, exposure to a therapeutic agent, exposure to a toxic agent or a potentially toxic agent, and/or exposure to a condition). Data in the data files also preferably includes values corresponding to level of proteins corresponding to the peptide fragmentation signatures found in a particular cell state.

[0110] In one embodiment, for a cell state determined by the differential expression of at least one protein, a data file corresponding to the cell state will minimally comprise data relating to the mass spectra observed after peptide fragmentation of a standard peptide diagnostic of the protein. Preferably, the data file will include a value corresponding to the level of the protein in a cell having the cell state. For example, a tumor cell state is associated with the overexpression of p53 (see, e.g., Kern, et al., 2001, Int. J. Oncol. 21(2): 243-9). The data file will comprise mass spectral data observed after fragmentation of a standard corresponding to a subsequence of p53. Preferably, the data file also comprises a value relating to the level of p53 in a tumor cell. The value may be expressed as a relative value (e.g., a ratio of the level of p53 in the tumor cell to the level of p53 in a normal cell) or as an absolute value (e.g., expressed in nM or as a % of total cellular proteins).

[0111] Preferably, the data files also include information relating to the presence or amount of a modified form of a target a polypeptide in at least one cell and to mass spectral data diagnostic of the modified form (i.e., peak data for a fragmented peptide internal standard which corresponds to the modified form). More preferably, the data files also comprise spectral data diagnostic of the unmodified form as well as data corresponding to the level of the unmodified form.

[0112] In one embodiment, data relating to ubiquitination sites and amounts of ubiquitination are stored in a database to create a proteome map of ubiquitinated proteins. Preferably, the database comprises a collection of data files relating to all ubiquitinated polypeptides in a particular cell type. The database preferably further comprises data relating to the origin of the cell, e.g., such as data relating to a patient from whom a cell was obtained. More preferably, the database comprises data relating to cells obtained from a plurality of patients. In one aspect, the database comprises data relating to the ubiquitination of a plurality of different cell types (e.g., cells from patients with a pathology, normal patients, cells at various stages of differentiation, and the like). In another aspect, data relating to ubiquitination patterns in cells obtained from patients with a neurological disease are stored in the database. For example, information relating to ubiquitination in cell samples from patients having any of Alzheimer's disease; amyotrophic lateral sclerosis; dementia; depression; Down's syndrome; Huntington's disease; peripheral neuropathy; multiple sclerosis; neurofibromatosis; Parkinson's disease; and schizophrenia, can be included in the database.

[0113] In a further embodiment, data relating to ubiquitination patterns in cells from patients with cancer are stored in the database, including, but not limited to patients with: adenocarcinoma; leukemia; lymphoma; melanoma; myeloma; sarcoma; teratocarcinoma; and, in particular, cancers of the adrenal gland; bladder; bone; bone marrow; brain; breast; cervix; gall bladder; ganglia; gastrointestinal; tract; heart, kidney; liver; lung; muscle; ovary; pancreas; parathyroid; prostate; salivary glands; skin; spleen; testes; thymus; thyroid; and uterus.

[0114] Additionally, data of ubiquitination patterns in cells from patients with an immune disorder may be included in the database. Such a disorder can include: acquired immunodeficiency syndrome (AIDS); Addison's disease; adult respiratory distress syndrome; allergies; ankylosing spondylitis; amyloidosis; anemia; asthma; atherosclerosis; autoimmune hemolytic anemia; autoimmune thyroiditis; bronchitis; cholecystitis; contact dermatitis; Crohn's disease; atopic dermatitis; dermatomyositis; diabetes mellitus; emphysema; episodic lymphopenia with lymphocytotoxins; erythroblastosis fetalis; erythema nodosum; atrophic gastritis; glomerulonephritis; Goodpasture's syndrome; gout; Graves' disease; Hashimoto's thyroiditis; hypereosinophilia; irritable bowel syndrome; myasthenia gravis; myocardial or pericardial inflammation; osteoarthritis; osteoporosis; pancreatitis; polymyositis; psoriasis; Reiter's syndrome; rheumatoid arthritis; scleroderma; Sjogren's syndrome; systemic anaphylaxis; systemic lupus erythematosus; systemic sclerosis; thrombocytopenic purpura; ulcerative colitis; uveitis; Werner syndrome; and viral, bacterial, fungal, parasitic, protozoal, and helminthic infections.

[0115] Data regarding ubiquitination in apoptotic cells and in pathologies associated with the misregulation of apoptosis also can be obtained using methods according to the invention.

[0116] In a further embodiment, data regarding ubiquitination in cardiac cells and cells from patients exhibiting a cardiac disease or at risk for a cardiac disease are obtained. In one aspect, the disease is an infarction or a condition relating to ischemia. In another aspect, the disease is cardiomyopathy.

[0117] Another aspect of the invention provides for kits for detecting and/or quantifying a polypeptide modification, such as ubiquitination. In one embodiment, the kit comprises a ubiquitin remnant specific binding partner and one or more components, including, but not limited to: a protease, preferably trypsin; a ubiquitinated molecule comprising known ubiquitination sites; acetonitrile; silica resin; heptafluorobutyric acid; urea (e.g., 8M urea); a sample plate for use with a mass spectrometer; a light-absorbent matrix; an ion exchange resin; software for analyzing mass spectra (e.g., such as SEQUEST); fused silica capillary tubing; and access to a computer memory comprising data files storing information relating to ubiquitination sites for a plurality of polypeptides for a plurality of different cells. Access may be in the form of a computer readable program product comprising the memory, or in the form of a URL and/or password for accessing an internet site for connecting a user to such a memory.

EXAMPLES

Example 1

[0118] Both polyclonal and monoclonal antibodies capable of recognizing the remnant of ubiquitin left from ubiquitinated proteins after digestion with the protease trypsin were generated. These antibodies were generated using a synthetic peptide library immunogen with the sequence CXXXXXXK(GG)XXXXXX, i.e., a Cysteine residue at the peptide amino-terminus, 6 "X" residues (X=any amino acid selected from all common amino acids excluding cysteine and tryptophan), a lysine residue ("K") that has been modified by addition of a Glycine-Glycine dipeptide to the epsilon-amino group of that lysine residue and 6 more "X" residues.

[0119] Polyclonal antibodies were generated by injecting rabbits with the peptide library immunogen described above conjugated either to keyhole limpet hemocyanin (KLH) or blue carrier protein. K(GG)-specific polyclonal antibodies from 6 rabbits: BL3415, BL3416, BL4933, BL4934, BL4935, BL4936.

[0120] BL4933, BL4935 were used as starting material for monoclonal antibody development. A monoclonal antibody from BL4933 was cloned and named recombinant antibody #3925 (D4A7A10). An additional monoclonal antibody was cloned from BL4935 (D24B6G9).

[0121] Table 1 shows the different monoclonal and polyclonal anti-ubiquitin remnant antibodies of the invention.

TABLE-US-00001 Monoclonal anti-Ubiquitin Polyclonal anti-Ubiquitin Remnant Antibodies Remnant Antibodies BL3415 BL3416 D4A7A10 BL4933 BL4934 D28B6G9 BL4935 BL4936

[0122] The heavy chain amino acid sequence of the D4A7A10 clone is provided in SEQ ID NO: 1. The light chain amino acid sequence of the D4A7A10 clone is provided in SEQ ID NO: 2. For the D4A7A10 clone (i.e., antibody #3925), using the CDR-defining rules set forth above, the CDR regions for the heavy and light chain are as follows:

TABLE-US-00002 Heavy Chain: CDR1 GFTISSNYYIYWV (SEQ ID NO: 3) CDR2 CIYGGSSGTTLYASWAKG (SEQ ID NO: 4) CDR3 DFRGADYSSYDRIWDTRLDL (SEQ ID NO: 5) Light Chain: CDR1 QSSENVYNKNWLS (SEQ ID NO: 6) CDR2 KASTLAS (SEQ ID NOL: 7) CDR3 AGDYGGTGDAFV (SEQ ID NO: 8)

[0123] The skilled artisan can readily determine the CDRs for the other antibodies disclosed herein including, without limitation, the antibody D24B6G9 cloned from BL4935.

Example 2

[0124] Characterization and Screening of Ubiquitin Tag Motif Antibodies. Anti-ubiquitin remnant peptide antibodies were characterized by differential peptide ELISA against antigen peptides CXXXXXXK(GG)XXXXXX (C02-1257) and control peptides CXXXXXXKXXXXXX(173-92A). All antibodies gave strong positive signals with antigen peptides and showed no binding with control peptides. Antibodies were validated by the peptide immunoprecipitation-MS methods described below by identifying ubiquitin-modified peptides in a trypsin-digested Jurkat cell lysate: antibodies passed this validation test when their use resulted in identification of most of the seven known ubiquitination sites in ubiquitin itself. These seven sites are shown in Table 2. Note that the some of the sites are represented in more than one peptide produced by trypsin digestion due to more than one trypsin cleavage sequence near the ubiquitinated site and/or due to more than one ubiquitinatable lysine residue in the peptide. For example, the ubiquitinated site at residue 48 is found in three trypic peptides (see Table 2).

TABLE-US-00003 TABLE 2 Known Ubiquitination Sites in Ubiquitin (where the asterisk following the lysing residue (i.e., K*) indicates the ubiquitinated residue) Residue Number Peptide Sequences 6 MQIFVK*TLTGK (SEQ ID NO: 9) 11 TLTGK*TITLEVEPSDTIENVK (SEQ ID NO: 10) TLTGK*TITLEVEPSDTIENVKAK (SEQ ID NO: 11) 27 TITLEVEPSDTIENVK*AKIQDKEGIPPDQQR (SEQ ID NO: 12) 29 AK*IQDKEGIPPDQQR (SEQ ID NO: 13) AK*IQDK*EGIPPDQQR (SEQ ID NO: 14) 33 IQDK*EGIPPDQQR (SEQ ID NO: 15) AKIQDK*EGIPPDQQR (SEQ ID NO: 16) AK*IQDK*EGIPPDQQR (SEQ ID NO: 17) 48 LIFAGK*QLEDGR (SEQ ID NO: 18) LIFAGK*QLEDGRTLSDYNIQK (SEQ ID NO: 19) LIFAGK*QLEDGRTLSDYNIQKESTLHLVLR (SEQ ID NO: 20) 63 TLSDYNIQK*ESTLHLVLR (SEQ ID NO: 21)

[0125] The antibodies of the invention were designed to recognize any peptide that contains ubiquitinated lysine residues regardless of surrounding peptide sequences. To illustrate the general context-independent recognition properties of one of these antibodies, the heat map shown in FIG. 2 shows the frequency of amino acids found with the BL4936 polyclonal antibody in a study of four mouse tissues. The studies were similar to the study described below in Example 3. Briefly, and by way of example, the cellular proteins are isolated from the tissue and digested with trypsin protease. Peptide purification was carried out, e.g., using Sep-Pak C₁₈ columns as described in Rush et al., U.S. Pat. No. 7,300,753). Following purification, peptides are lyophilized and then resuspended in MOPS buffer (50 mM MOPS/NaOH pH 7.2, 10 mM Na₂HPO₄, 50 mM NaCl) and insoluble material removed by centrifugation at 12,000×g for 10 minutes. The anti-ubiquitin remnant antibodies of the invention were coupled non-covalently to protein G agarose beads (Roche) at 4 mg/ml beads overnight at 4° C. After coupling, antibody-resin was washed twice with PBS and three times with MOPS buffer. Immobilized antibody (40 μl, 160 μg) was added as a 1:1 slurry in MOPS IP buffer to the solubilized peptide fraction, and the mixture was incubated overnight at 4° C. The immobilized antibody beads were washed three times with MOPS buffer and twice with ddH₂O. Peptides were eluted twice from beads by incubation with 50 μl of 0.15% TFA for 15 minutes each, and the fractions were combined and analyzed by LC-MS/MS mass spectrometry.

[0126] Altogether 1458 non-redundant peptides were included in the frequency map shown in FIG. 2. The map clearly shows there are no strongly preferred amino acids at least seven residues to the amino-terminal side of K(GG) modification sites (-7 to -1 in FIG. 2) or at least seven residues to the carboxyl-terminal side of K(GG) modification sites (1 to 7 in FIG. 2).

Example 3

[0127] Numerous experiments were performed using the isolated antibodies of the invention in the methods described in U.S. Pat. Nos. 7,198,896 and 7,300,753. Table 3 lists some of these experiments performed and the number of ubiquitinated peptides observed (both redundant and non-redundant) in each of these experiments.

TABLE-US-00004 TABLE 3 Expt. Non- No. Antibody Cell/Tissue Type Treatment 1 Treatment 2 Redundant Redundant 3114 BL4936 mouse heart 447 332 3115 BL4936 mouse liver 790 591 3116 BL4936 Embryo mouse brain 662 548 3117 BL4936 Adult mouse brain 735 565 3573 BL4936 rat brain sham mock surgery 738 553 3574 BL4936 rat brain sham mock surgery 833 618 3575 BL4936 rat brain ischemia ischemia Reperfusion 760 554 30' R 3576 BL4936 rat brain ischemia ischemia Reperfusion 809 580 30' R 3577 BL4936 rat brain ischemia ischemia Reperfusion 741 551 24h R 3578 BL4936 rat brain ischemia ischemia Reperfusion 773 567 24h R 3970 BL4936 rat brain sham untreated 693 499 3971 BL4936 rat brain ischemia Reperfusion 829 604 30' R 3972 BL4936 rat brain ischemia Reperfusion 816 620 24h R 4120 BL4934 AD control untreated 413 271 4121 BL4934 AD control untreated 382 249 4122 BL4934 AD control untreated 388 265 4123 BL4934 AD control untreated 488 326 4124 BL4934 AD control untreated 406 278 4125 BL4934 AD control untreated 478 321 4126 BL4934 AD+/- untreated 453 324 4127 BL4934 AD+/- untreated 508 343 4128 BL4934 AD+/- untreated 384 258 4129 BL4934 AD+/- untreated 265 181 5338 BL4934 Jurkat pervanadate calyculin 217 173 5339 BL4936 Jurkat pervanadate calyculin 202 161 5566 BL4934 MKN-45 Su11274 668 394 5567 BL4934 MKN-45 Su11274 565 353 5642 BL4933 H2228 silac1 DMSO 615 408 5643 BL4933 H2228 silac2 inhibitor 556 326 5644 BL4933 H2228 silac3 inhibitor 463 298 5645 BL4933 H2228 silac4 inhibitor 415 272 5712 D24B6G9 Jurkat pervanadate calyculin 137 105 5972 BL4934 H3122 Silac inh1 inhibitor 353 200 5973 BL4934 H3122 Silac inh2 inhibitor 247 185 5974 BL4934 H3122 Silac inh3 inhibitor 391 245 6090 BL4934 H2228 silac Dana inhibitor 193 135 Farber 6093 BL4934 H3122 Silac Dana inhibitor 178 140 Farber 6131 BL4934 H1703 normal 978 691 6362 D24B6G9 Jurkat pervanadate calyculin 431 283 6586 BL4935 U266 control 793 539 6587 BL4935 U266 MG132 791 522 6588 BL4935 U266 MG132 1074 867 6589 BL4935 H929 control 1265 764 6590 BL4935 H929 MG132 712 468 6591 BL4935 H929 MG132 551 467 6846 BL4935 H1703 735 484 6847 BL4935 H1703 1143 841 6916 BL4935 RAW 264.7 normal 1366 736 6917 BL4935 RAW 264.7 LPS 1396 746 6918 BL4935 RAW 264.7 LPS 1424 771 6919 BL4935 RAW 264.7 MG132 1473 871 6939 D4A7A10 Jurkat pervanadate calyculin 286 240 6941 BL4933 Jurkat pervanadate calyculin 130 102 8149 D4A7A10 Jurkat calyculin pervanadate 613 445 8158 D4A7A10 mouse muscle untreated 886 651 8159 D4A7A10 mouse spleen untreated 1355 1033 8160 D4A7A10 mouse testis untreated 1096 872 8161 D4A7A10 mouse thymus untreated 827 623 8241 D4A7A10 LNCaP control 940 801 8242 D4A7A10 LNCaP AAG 978 826 8243 D4A7A10 LNCaP AAG 561 474 8244 D4A7A10 LNCaP Geldanamycin 874 747 8245 D4A7A10 LNCaP Geldanamycin 665 569 8246 D4A7A10 LNCaP Velcade 970 808 8247 D4A7A10 LNCaP Velcade 1056 907

[0128] The exemplary results shown below in Table 4 correspond to experiment number 3115 in Table 3. In the experiment, cellular proteins from 500 mg of mouse liver were denatured with urea, reduced with dithiothreitol, alkylated with iodoacetamide digested with the protease trypsin. The resulting peptides were separated from other cellular materials by reversed-phased solid phase extraction, then lyophilized and resuspended. Peptides containing the K(GG) modification were separated from other peptides by treatment with the immobilized polyclonal anti-ubiquitin remnant antibody BL4936. The BL4936 associated beads were washed, and bound K(GG)-peptides were eluted with dilute trifluouroacetic acid. The peptides were concentrated and then analyzed by liquid chromatography-tandem mass spectrometry LC-MS. See for example, U.S. Pat. Nos. 7,198,896 and 7,300,753, the entire disclosures of which are incorporated by reference.

TABLE-US-00005 TABLE 4 Known and Novel Ubiquitination Sites Found in One Analysis of Proteins from Mouse Liver Ubiquitinated Protein Residue SEQ ID Row Protein Type Name Number Peptide Sequence NO: 1 Unassigned ADRM1 %34 MSLK*GTTVTPDKRK 22 2 Unassigned ADRM1 %34 MSLK*GTTVTPDKR 23 3 Unassigned ADRM1 %34 MSLK*GTTVTPDK 24 4 Unassigned ADRM1 %34 M#SLK*GTTVTPDKR 25 5 Unassigned ADRM1 %34 M#SLK*GTTVTPDKRK 26 6 Receptor, GLT1 %517 MQEDIEMTK*TQSIYDDKNHR 27 channel, transporter or cell surface protein 7 Chromatin, H1D; H1C %45; %46 KASGPPVSELITK*AVAASK 28 DNA-binding, DNA repair or DNA replication protein 8 Chromatin, H1D; H1E; %63; %63; K*ALAAAGYDVEK 29 DNA-binding, H1C; H1T %64; %66 DNA repair or DNA replication protein 9 Chromatin, H1D; H1E; %63; %63; K*ALAAAGYDVEKNNSR 30 DNA-binding, H1C; H1T %64; %66 DNA repair or DNA replication protein 10 Chromatin, H1D; H1E; %74; %74; ALAAAGYDVEK*NNSR 31 DNA-binding, H1C; H1T %75; %77 DNA repair or DNA replication protein 11 Chromatin, H1E %45 KTSGPPVSELITK*AVAASK 32 DNA-binding, DNA repair or DNA replication protein 12 Chromatin, H1E %45 TSGPPVSELITK*AVAASK 33 DNA-binding, DNA repair or DNA replication protein 13 Chromatin, H2A.1; %119; VTIAQGGVLPNIQAVLLPK*KTESHHK 34 DNA-binding, H2AO; H2AE %118; DNA repair or %119 DNA replication protein 14 Chromatin, H2A.1; %120; 119; VTIAQGGVLPNIQAVLLPKK*TESHHK 35 DNA-binding, H2AO; H2AE %120 DNA repair or DNA replication protein 15 Chromatin, H2A.1; %119; VTIAQGGVLPNIQAVLLPK*K 36 DNA-binding, H2AX; %118; 119; DNA repair or HIST2H2AB; 119; %118; DNA HIST2H2AC; %119; replication H2AO; %119; protein H2A.4; %119; H2AE; H2AL; %119 H2AFJ 16 Chromatin, H2A.1; %120; VTIAQGGVLPNIQAVLLPKK* 37 DNA-binding, H2AX; %119; 120; DNA repair or HIST2H2AB; 120; 119; DNA HIST2H2AC; 120; %120; replication H2AO; %120; protein H2A.4; %120 H2AE; H2AL; H2AFJ 17 Unassigned H2AE %120 VTIAQGGVLPNIQAVLLPKK*TESHH 38 KPK 18 Chromatin, H2AFJ %119 VTIAQGGVLPNIQAVLLPK*KTESQK 39 DNA-binding, DNA repair or DNA replication protein 19 Chromatin, H2AFJ %120 VTIAQGGVLPNIQAVLLPKK*TESQK 40 DNA-binding, DNA repair or DNA replication protein 20 Chromatin, H2AFY %116 GVTIASGGVLPNIHPELLAK*K 41 DNA-binding, DNA repair or DNA replication protein 21 Chromatin, H2AFY %116 GVTIASGGVLPNIHPELLAK*KR 42 DNA-binding, DNA repair or DNA replication protein 22 Chromatin, H2AFY %117 GVTIASGGVLPNIHPELLAKK*R 43 DNA-binding, DNA repair or DNA replication protein 23 Chromatin, H2AL %120 VTIAQGGVLPNIQAVLLPKK*TETHHK 44 DNA-binding, DNA repair or DNA replication protein 24 Chromatin, H2AX %119 K*SSATVGPK 45 DNA-binding, DNA repair or DNA replication protein 25 Chromatin, H2AX; %118; 119 LLGGVTIAQGGVLPNIQAVLLPK*K 46 DNA-binding, HIST2H2AB DNA repair or DNA replication protein 26 Chromatin, H2AX; %118; 119 NDEELNKLLGGVTIAQGGVLPNIQA 47 DNA-binding, HIST2H2AB VLLPK*K DNA repair or DNA replication protein 27 Chromatin, H2B; H2B1D; %120; AVTK*YTSSK 48 DNA-binding, H2B1A; %120; DNA repair or H2B1N; %122; DNA H2B2E; %121; replication H2B1H; %121; protein H2B1C; %121; Hist3h2ba %121; %121 28 Chromatin, H2B; H2B1D; %46; %46; VLK*QVHPDTGISSK 49 DNA-binding, H2B1A; %48; %47; DNA repair or H2B1N; %47; %47; DNA H2B2E; %47; %47; replication H2B1L; %47 protein H2B1H; H2B1C; Hist3h2ba 29 Chromatin, H2B; H2B1D; %116; HAVSEGTK*AVTK 50 DNA-binding, H2B1A; %116; DNA repair or H2B1N; %118; DNA H2B2E; %117; replication H2B1L; %117; protein H2B1H; %117; H2B1C; %117; Hist3h2ba %117; %117 30 Chromatin, H2B1L %121 AVTK*YTSAK 51 DNA-binding, DNA repair or DNA replication protein 31 Unassigned HIST2H2AB; 119; 119; VTIAQGGVLPNIQAVLLPK*KTESHK 52 HIST2H2AC; %119 H2A.4 32 Unassigned HIST2H2AB; 125; 125; VTIAQGGVLPNIQAVLLPKKTESHK* 53 HIST2H2AC; %125 H2A.4 33 Chaperone HSC70; 507; 509; ITITNDK*GR 54 HSPA1L; 510; 507; HSPA2; %507 HSP70-2; HSP70 34 Ubiquitin NEDD8 %48 LIYSGK*QMNDEK 55 conjugating system 35 Unassigned RPS20 %8 DTGK*TPVEPEVAIHR 56 36 Unknown SPG20 %360 SSHPSEPPK*EASGTDVR 57 function 37 Protein Titin %30428 EAFSSVIIK*EPQIEPTADLTGITNQLI 58 kinase, TCK Ser/Thr (non- receptor) 38 Cytoskeletal TUBA1B; %370; 370; VGINYQPPTVVPGGDLAK*VQR 59 protein TUBA3D; 370; 370; TUBA4A; 370; 370; TUBA1A; 369 TUBA1C; TUBA8; TUBA3C 39 Ubiquitin UBA52; 11; %11 TLTGK*TITLEVEPSDTIENVK 60 conjugating ubiquitin system 40 Ubiquitin UBA52; 6; %6; 6 MQIFVK*TLTGK 61 conjugating ubiquitin; system LOC388720 41 Ubiquitin UBA52; 29; %29; 29; AK*IQDKEGIPPDQQR 62 conjugating ubiquitin; 106 system LOC388720; Gm7866 42 Ubiquitin UBA52; 29, 33; %29, AK*IQDK*EGIPPDQQR 63 conjugating ubiquitin; %33; 29, 33;

system LOC388720; 106, 110 Gm7866 43 Ubiquitin UBA52; 33; %33; 33; IQDK*EGIPPDQQR 64 conjugating ubiquitin; 110 system LOC388720; Gm7866 44 Ubiquitin UBA52; 33; %33; 33; AKIQDK*EGIPPDQQR 65 conjugating ubiquitin; 110 system LOC388720; Gm7866 45 Ubiquitin UBA52; 48; %48; 48; LIFAGK*QLEDGR 66 conjugating ubiquitin; 125 system LOC388720; Gm7866 46 Ubiquitin UBA52; 48; %48; 48; LIFAGK*QLEDGRTLSDYNIQK 67 conjugating ubiquitin; 125 system LOC388720; Gm7866 47 Ubiquitin UBA52; 48; %48; 48; LIFAGK*QLEDGRTLSDYNIQKESTL 68 conjugating ubiquitin; 125 HLVLR system LOC388720; Gm7866 48 Ubiquitin UBA52; 63; %63; 63; TLSDYNIQK*ESTLHLVLR 69 conjugating ubiquitin; 63; 140 system LOC388720; OTTMUSG00 000001634; Gm7866 49 Mitochondrial 1190003J15Rik 67 CPGLLTPSQIKPGTYK*LFFDTER 70 protein 50 Unassigned 1300002K09Rik 223 SM#LEAHQAKHVK*QLLSKPR 71 51 Adaptor/ 14-3-3 eta; 49; 49; 49; NLLSVAYK*NVVGAR 72 scaffold 14-3-3 50 gamma; 14- 3-3 zeta; 14- 3-3 beta 52 Enzyme, misc. 1-Cys PRX 198 KGESVM#VVPTLSEEEAK*QCFPK 73 53 Enzyme, misc. 1-Cys PRX 198 KGESVMVVPTLSEEEAK*QCFPK 74 54 Enzyme, misc. 1-Cys PRX 208 GVFTK*ELPSGK 75 55 Receptor, ABCA3 503 TVVGK*EEEGSDPEK 76 channel, transporter or cell surface protein 56 Receptor, ABCA3 503, 512 TVVGK*EEEGSDPEK*ALR 77 channel, transporter or cell surface protein 57 Receptor, ABCA3 1620 SEGK*QDALEEFK 78 channel, transporter or cell surface protein 58 Unassigned ABCB11 935 EILEK*AGQITNEALSNIR 79 59 Unassigned ABCB11 935 MLTGFASQDKEILEK*AGQITNEAL 80 SNIR 60 Unassigned ABCB11 935 M#LTGFASQDKEILEK*AGQITNEAL 81 SNIR 61 Receptor, ABCC2 491 KIQVQNM#K*NK 82 channel, transporter or cell surface protein 62 Receptor, ABCC2 491 IQVQNMK*NK 83 channel, transporter or cell surface protein 63 Adhesion or ABHD2 57 FLLK*SCPLLTK 84 extracellular matrix protein 64 Translation AC078817.18- 136; 136 GKYK*EETIEK 85 1; RPL26 65 Enzyme, misc. ACAA1b; 292; 292 RSK*AEELGLPILGVLR 86 ACAA1 66 Enzyme, misc. ACOX1 488 IQPQQVAVWPTLVDINSLDSLTEAY 87 K*LR 67 Enzyme, misc. ACSL5 361 VYDK*VQNEAK 88 68 Enzyme, misc. ACSL5 616 NQCVK*EAILEDLQK 89 69 Enzyme, misc. ACSL5 675 FFQTQIK*SLYESIEE 90 70 Cytoskeletal ACTG2; 193; 193; DLTDYLMK*ILTER 91 protein ACTC1; 193; 191; ACTA1; 192; 195 ACTB; ACTBL2; ACTG1 71 Cytoskeletal ACTG2; 328; 328; EITALAPSTM#K*IK 92 protein ACTC1; 328; 326; ACTA1; 330 ACTB; ACTG1 72 Enzyme, misc. ADCY3 297 HVADEMLKDMKK* 93 73 Mitochondrial ADH1C 40 IK*MVATGVCR 94 protein 74 Mitochondrial ADH1C 105 ICK*HPESNFCSR 95 protein 75 Mitochondrial ADH1C 169 IDGASPLDK*VCLIGCGFSTGYGSA 96 protein VK 76 Mitochondrial ADH1C 186 IDGASPLDKVCLIGCGFSTGYGSAV 97 protein K*VAK 77 Mitochondrial ADH1C 316 TWK*GAIFGGFK 98 protein 78 Mitochondrial ADH1C 339 LVADFMAK*K 99 protein 79 Kinase (non- ADK 110 AATFFGCIGIDK*FGEILK 100 protein) 80 Kinase (non- ADK 255 EQGFETK*DIK 101 protein) 81 Kinase (non- ADK 357 TGCTFPEK*PDFH 102 protein) 82 Enzyme, misc. AKR1C1 225 EK*QWVDQSSPVLLDNPVLGSMAK 103 83 Enzyme, misc. AKR1C1 312 YISGSSFK*DHPDFPFWDEY 104 84 Enzyme, misc. ALAD 87 VPK*DEQGSAADSEDSPTIEAVR 105 85 Enzyme, misc. ALAD 87 CVLIFGVPSRVPK*DEQGSAADSED 106 SPTIEAVR 86 Enzyme, misc. ALAD 184 AALLK*HGLGNR 107 87 Receptor, albumin 460 VGTK*CCTLPEDQR 108 channel, transporter or cell surface protein 88 Unassigned ALDH16A1 603 RK*PVLTSQLER 109 89 Enzyme, misc. ALDH1A1 434 ANNTTYGLAAGLFTK*DLDK 110 90 Enzyme, misc. ALDH1A1 434 RANNTTYGLAAGLFTK*DLDK 111 91 Enzyme, misc. ALDH1A2 338 IFVEESIYEEFVK* 112 92 Unassigned Aldh1a7 435 ANNTTYGLAAGVFTK*DLDK 113 93 Unassigned Aldh1a7 499 TVAMQISQK*NS 114 94 Enzyme, misc. Aldh1a7; 91; 90 LLNK*LADLMERDR 115 ALDH1A1 95 Enzyme, misc. Aldh1a7; 91; 90 LLNK*LADLMER 116 ALDH1A1 96 Enzyme, misc. Aldh1a7; 255; 254 LIK*EAAGK 117 ALDH1A1 97 Enzyme, misc. Aldh1a7; 378; 377 WGNK*GFFVQPTVFSNVTDEM#R 118 ALDH1A1 98 Enzyme, misc. Aldh1a7; 378; 377 WGNK*GFFVQPTVFSNVTDEMR 119 ALDH1A1 99 Enzyme, misc. Aldh1a7; 398; 397 IAK*EEIFGPVQQIMK 120 ALDH1A1 100 Enzyme, misc. ALDH3A2 296 LQSLLK*GQK 121 101 Enzyme, misc. ALDH7A1 424 FQDEEEVFEWNNEVK*QGLSSSIFTK 122 102 Enzyme, misc. ALDOB 47 IK*VENTEENRR 123 103 Enzyme, misc. ALDOB 107 GIVVGIK*LDQGGAPLAGTNK 124 104 Enzyme, misc. ALDOB 107 GIVVGIK*LDQGGAPLAGTNKETTIQ 125 GLDGLSER 105 Enzyme, misc. ALDOB 120 LDQGGAPLAGTNK*ETTIQGLDGLS 126 ER 106 Enzyme, misc. ALDOB 329 ATQEAFMK*R 127 107 Adhesion or AMFR 573 FSK*SADER 128 extracellular matrix protein 108 Adhesion or AMFR 600 FLNK*SSEDDGASER 129 extracellular matrix protein 109 Calcium- ANXA6 477 AINEAYK*EDYHK 130 binding protein 110 Unassigned Apoc1 60 AAIEHIK*QK 131 111 Unassigned ApoE 105 LGK*EVQAAQAR 132 112 Unassigned ApoE 252 SK*MEEQTQQIR 133 113 Unassigned APOL3 232 GMK*EVLDQSGPR 134 114 Unassigned Apol9b 184 IVNK*IPQATR 135 115 Enzyme, misc. ARG1 26 GGVEK*GPAALR 136 116 Enzyme, misc. ARG1 205 YFSMTEVDK*LGIGK 137 117 Unassigned ARIH1 314 QFCFNCGENWHDPVK*CK 138 118 Enzyme, misc. ASL 43 HLWNVDVQGSK*AYSR 139 119 Endoplasmic ASS1 112 EGAK*YVSHGATGK 140 reticulum or golgi 120 Endoplasmic ASS1 121 YVSHGATGK*GNDQVR 141 reticulum or golgi

121 Endoplasmic ASS1 340 HCIQK*SQERVEGK 142 reticulum or golgi 122 Endoplasmic ASS1 340 HCIQK*SQER 143 reticulum or golgi 123 Chromatin, ASXL2 325 KVELWK*EQFFENYYGQSSGLSLE 144 DNA-binding, DSQK DNA repair or DNA replication protein 124 Unknown AUP1 250 VQQLVAK*ELGQIGTR 145 function 125 Unknown BAT3 56 EHIAASVSIPSEK*QR 146 function 126 Unassigned BC066028 365, 368 THGRAK*SYK*CGECGK 147 127 Transcriptional BCoR-like 1; 1491; 1464 LIVNK*NAGETLLQR 148 regulator BCoR 128 Enzyme, misc. BHMT; 283; 274 WDIQK*YAR 149 BHMT2 129 Ubiquitin BRAP 380 LVASK*TDGK 150 conjugating system 130 Unassigned C4orf34 83 GSSLPGK*PSSPHSGQDPPAPPVD 151 131 Enzyme, misc. CA3 39 DIK*HDPSLQPWSASYDPGSAK 152 132 Enzyme, misc. CA3 57 DIKHDPSLQPWSASYDPGSAK*TIL 153 NNGK 133 Endoplasmic catalase 242 TDQGIK*NLPVGEAGR 154 reticulum or golgi 134 Enzyme, misc. CBS 386 FLSDK*WMLQK 155 135 Chaperone CCT-alpha 126 LACK*EAVR 156 136 Chaperone CCT-alpha 541 DDK*HGSYENAVHSGALDD 157 137 Chaperone CCT-theta 533 VDQIIMAKPAGGPK*PPSGKKDWD 158 DDQND 138 Chaperone CCT-theta 538 PAGGPKPPSGK*KDWDDDQND 159 139 Chaperone CCT-theta 539 VDQIIMAKPAGGPKPPSGKK*DWD 160 DDQND 140 Unassigned CHIC1 179 SIQK*LLEWENNR 161 141 Unknown CIRH1A 642, 645, RTTHGFK*MSK*IYK* 162 function 648 142 Cytoskeletal claudin 3 216 STGPGTGTGTAYDRK*DYV 163 protein 143 Unassigned CLIC4 202 LHIVKVVAK* 164 144 Vesicle protein CLTC 629 AHIAQLCEK*AGLLQR 165 145 Vesicle protein CLTC 1450 AVNYFSK*VK 166 146 Vesicle protein CLTC 1452 VK*QLPLVKPYLR 167 147 Mitochondrial CPS1 307 EPLFGISTGNIITGLAAGAK*SYK 168 protein 148 Mitochondrial CPS1 310 SYK*MSMANR 169 protein 149 Mitochondrial CPS1 560 QLFSDKLNEINEK*IAPSFAVESMED 170 protein ALK 150 Mitochondrial CPS1 772 TSACFEPSLDYMVTK*IPR 171 protein 151 Mitochondrial CPS1 1100 SIFSAVLDELK*VAQAPWK 172 protein 152 Mitochondrial CPS1 1183 EVEMDAVGK*EGR 173 protein 153 Mitochondrial CPS1 1269 SFPFVSK*TLGVDFIDVATK 174 protein 154 Enzyme, misc. CPT1A 195 YLESVRPLMK*EGDFQR 175 155 Enzyme, misc. CRAD2 64 VLAACLTEK*GAEQLR 176 156 Enzyme, misc. CRAD2 224 LSHSIEK*LWDQTSSEVKEVYDKNF 177 LDSYIK 157 Cell cycle CTH 47 AVVLPISLATTFK*QDFPGQSSGFE 178 regulation YSR 158 Cell cycle CTH 72 NCLEK*AVAALDGAK 179 regulation 159 Adhesion or CTNNB1 671 M#SEDKPQDYK*K 180 extracellular matrix protein 160 Adhesion or CTNNB1 671 MSEDKPQDYK*K 181 extracellular matrix protein 161 Adaptor/ CTNND1 517 MEIVDHALHALTDEVIIPHSGWERE 182 scaffold PNEDCK*PR 162 Adaptor/ CTNND1 517 M#EIVDHALHALTDEVIIPHSGWER 183 scaffold EPNEDCK*PR 163 Adaptor/ CTNND1 710 SALRQEK*ALSAIAELLTSEHER 184 scaffold 164 Receptor, Cx32 244 LSPEYK*QNEINK 185 channel, transporter or cell surface protein 165 Receptor, Cx32 276 SPGTGAGLAEK*SDR 186 channel, transporter or cell surface protein 166 Receptor, Cx32 276 RSPGTGAGLAEK*SDR 187 channel, transporter or cell surface protein 167 Adhesion or CXADR 271 YEK*EVHHDIR 188 extracellular matrix protein 168 Unassigned CYB5A 38 VYDLTK*FLEEHPGGEEVLR 189 169 Enzyme, misc. CYB5R3 240 LWYTVDK*APDAWDYSQGFVNEE 190 M#IR 170 Enzyme, misc. CYP1A1; 97; 94 IGSTPVVVLSGLNTIK*QALVR 191 CYP1A2 171 Enzyme, misc. CYP1A2 250 YLPNPALK*R 192 172 Enzyme, misc. CYP1A2 276 TVQEHYQDFNK*NSIQDITSALFK 193 173 Enzyme, misc. CYP1A2 294 HSENYK*DNGGLIPEEK 194 174 Enzyme, misc. CYP1A2 401 DTSLNGFHIPK*ER 195 175 Enzyme, misc. Cyp2a12 250 DSHKLEDFMIQK*VK 196 176 Enzyme, misc. Cyp2a12 252 VK*QNQSTLDPNSPR 197 177 Endoplasmic CYP2A7 32 LSGK*LPPGPTPLPFVGNFLQLNTE 198 reticulum or QM#YNSLM#K golgi 178 Endoplasmic CYP2A7 32 LSGK*LPPGPTPLPFVGNFLQLNTE 199 reticulum or QMYNSLM#K golgi 179 Endoplasmic CYP2A7 32 LSGK*LPPGPTPLPFVGNFLQLNTE 200 reticulum or QMYNSLMK golgi 180 Endoplasmic CYP2A7; 239; 239 HLPGPQQQAFK*ELQGLEDFITK 201 reticulum or Cyp2a5 golgi 181 Endoplasmic CYP2A7; 342; 342 NRQPK*YEDR 202 reticulum or Cyp2a5 golgi 182 Endoplasmic CYP2A7; 348; 348 MK*MPYTEAVIHEIQR 203 reticulum or Cyp2a5 golgi 183 Endoplasmic CYP2A7; 409; 409 FFSNPK*DFNPK 204 reticulum or Cyp2a5 golgi 184 Endoplasmic CYP2A7; 250; 250; 35 ELQGLEDFITK*K 205 reticulum or Cyp2a5; golgi Cyp2a21-ps 185 Enzyme, misc. CYP2B1; 346; 345; TK*MPYTDAVIHEIQR 206 Cyp2b9; 345 Cyp2b13 186 Enzyme, misc. CYP2C19; 432; 331; KSDYFMPFSTGK*R 207 Cyp2c29; 432; 432; CYP2C9; 432 Cyp2c54; Cyp2c50 187 Enzyme, misc. CYP2C19; 432; 331; SDYFMPFSTGK*R 208 Cyp2c29; 432; 432; CYP2C9; 432 Cyp2c54; Cyp2c50 188 Enzyme, misc. CYP2C19; 84; 84 KPTVVLHGYEAVK*EALVDHGEEFA 209 Cyp2c50 GR 189 Unassigned Cyp2c29 298 GTTVITSLSSVLHDSK*EFPNPEM# 210 FDPGHFLNGNGNFK 190 Enzyme, misc. Cyp2c39 399 GTTVVTSLTSVLHDSK*EFPNPELF 211 DPGHFLDANGNFK 191 Enzyme, misc. Cyp2c39; 270; 169; DFIDYYLIK*QK 212 Cyp2c29; 270 CYP2C9 192 Enzyme, misc. Cyp2c40 375 YIDLGPNGVVHEVTCDTK*FR 213 193 Enzyme, misc. Cyp2c40; 110; 110 GK*GIGFSHGNVWK 214 LOC100048323 194 Enzyme, misc. Cyp2c40; 154; 154 VQEEAQWLM#K*ELKK 215 LOC100048323 195 Enzyme, misc. Cyp2c40; 154; 154 VQEEAQWLMK*ELKK 216 LOC100048323 196 Enzyme, misc. Cyp2c40; 154; 154 VQEEAQWLMK*ELK 217 LOC100048323 197 Enzyme, misc. Cyp2c40; 154; 154 VQEEAQWLM#K*ELK 218 LOC100048323 198 Enzyme, misc. Cyp2c40; 157; 157 VQEEAQWLM#KELK* 219 LOC100048323 199 Enzyme, misc. Cyp2c40; 157; 157 VQEEAQWLMKELK*K 220 LOC100048323

200 Enzyme, misc. Cyp2c40; 249; 249 IK*EHEESLDVTNPR 221 LOC100048323 201 Enzyme, misc. Cyp2c54 84 KPTVVLHGYEAVK*EALVDHGDVF 222 AGR 202 Unassigned Cyp2c70 234 FLK*DVTQQK 223 203 Unassigned Cyp2c70 234 FLK*DVTQQKK 224 204 Unassigned Cyp2c70 252 HQK*SLDLSNPQDFIDYFLIK 225 205 Unassigned Cyp2d10 414 GSILIPNM#SSVLKDETVWEK*PLR 226 206 Enzyme, misc. CYP2D2 414 GTTLIPNLSSVLKDETVWEK*PLR 227 207 Unassigned Cyp2d40 252 GTTLICNLSSVLKDETVWEK*PLR 228 208 Endoplasmic CYP2E1 59 SLTK*LAK 229 reticulum or golgi 209 Endoplasmic CYP2E1 84 IVVLHGYK*AVK 230 reticulum or golgi 210 Endoplasmic CYP2E1 84 RIVVLHGYK*AVK 231 reticulum or golgi 211 Endoplasmic CYP2E1 87 AVK*EVLLNHKNEFSGR 232 reticulum or golgi 212 Endoplasmic CYP2E1 94 EVLLNHK*NEFSGR 233 reticulum or golgi 213 Endoplasmic CYP2E1 110 GDIPVFQEYK*NK 234 reticulum or golgi 214 Endoplasmic CYP2E1 112 NK*GIIFNNGPTWK 235 reticulum or golgi 215 Endoplasmic CYP2E1 123 GIIFNNGPTWK*DVR 236 reticulum or golgi 216 Endoplasmic CYP2E1 140 DWGM#GK*QGNEAR 237 reticulum or golgi 217 Endoplasmic CYP2E1 140 DWGMGK*QGNEAR 238 reticulum or golgi 218 Endoplasmic CYP2E1 159 EAHFLVEELK*K 239 reticulum or golgi 219 Endoplasmic CYP2E1 162 TK*GQPFDPTFLIGCAPCNVIADILF 240 reticulum or NK golgi 220 Endoplasmic CYP2E1 255 AKEHLK*SLDINCPR 241 reticulum or golgi 221 Endoplasmic CYP2E1 255 EHLK*SLDINCPR 242 reticulum or golgi 222 Endoplasmic CYP2E1 275 DVTDCLLIEMEK*EK 243 reticulum or golgi 223 Endoplasmic CYP2E1 428 YSDYFK*AFSAGK 244 reticulum or golgi 224 Endoplasmic CYP2E1 428 YSDYFK*AFSAGKR 245 reticulum or golgi 225 Endoplasmic CYP2E1 467 SLVDPK*DIDLSPVTIGFGSIPR 246 reticulum or golgi 226 Receptor, CYP3A4 35 K*QGIPGPTPLPFLGTVLNYYK 247 channel, transporter or cell surface protein 227 Receptor, CYP3A4 380 FCKK*DVELNGVYIPK 248 channel, transporter or cell surface protein 228 Receptor, CYP3A4 425 ENK*GSIDPYLYMPFGIGPR 249 channel, transporter or cell surface protein 229 Receptor, CYP3A4 425 ENK*GSIDPYLYM#PFGIGPR 250 channel, transporter or cell surface protein 230 Receptor, CYP3A4 425 FSKENK*GSIDPYLYMPFGIGPR 251 channel, transporter or cell surface protein 231 Receptor, CYP3A4 477 VMQNFSFQPCQETQIPLK*LSR 252 channel, transporter or cell surface protein 232 Receptor, CYP3A43; 421; 422; FSK*ENK 253 channel, CYP3A5; 422; 558; transporter or CYP3A7; 657; 422; cell surface UVRAG; 422 protein KIAA1802; Cyp3a44; CYP3A4 233 Unassigned Cyp3a44 422 FSK*ENKGSIDPYVYLPFGIGPR 254 234 Unassigned Cyp3a44 488 QGILQPEK*PIVLK 255 235 Unassigned Cyp3a44 493 QGILQPEKPIVLK*VVPR 256 236 Receptor, Cyp3a44; 116; 116; 16 EFGPVGIMSK*AISISKDEEWKR 257 channel, CYP3A4; transporter or LOC673748 cell surface protein 237 Receptor, CYP3A5; 96; 96 NVLVK*ECFSVFTNRR 258 channel, CYP3A4 transporter or cell surface protein 238 Receptor, CYP3A5; 141; 141 ALLSPTFTSGK*LK 259 channel, CYP3A4 transporter or cell surface protein 239 Receptor, CYP3A5; 488; 488 QGLLQPEK*PIVLK 260 channel, CYP3A4 transporter or cell surface protein 240 Enzyme, misc. CYP3A5; 35; 35 K*QGIPGPKPLPFLGTVLNYYK 261 CYP3A7 241 Enzyme, misc. CYP3A5; 42; 42 QGIPGPK*PLPFLGTVLNYYK 262 CYP3A7 242 Receptor, CYP3A5; 96; 96; 96 NVLVK*ECFSVFTNR 263 channel, CYP3A7; transporter or CYP3A4 cell surface protein 243 Enzyme, misc. CYP3A5; 158; 158; LKEM#FPVIEQYGDILVK*YLR 264 CYP3A7; 158 Cyp3a44 244 Enzyme, misc. CYP3A5; 158; 158; EM#FPVIEQYGDILVK*YLR 265 CYP3A7; 158 Cyp3a44 245 Enzyme, misc. CYP3A5; 158; 158; LKEMFPVIEQYGDILVK*YLR 266 CYP3A7; 158 Cyp3a44 246 Receptor, CYP3A5; 143; 143; LK*EM#FPVIEQYGDILVK 267 channel, CYP3A7; 143; 143 transporter or Cyp3a44; cell surface CYP3A4 protein 247 Receptor, CYP3A5; 143; 143; LK*EMFPVIEQYGDILVK 268 channel, CYP3A7; 143; 143 transporter or Cyp3a44; cell surface CYP3A4 protein 248 Receptor, CYP3A5; 250; 250; DSIEFFK*K 269 channel, CYP3A7; 250; 250 transporter or Cyp3a44; cell surface CYP3A4 protein 249 Receptor, CYP3A7; 59; 59 GLWK*FDMECYEK 270 channel, CYP3A4 transporter or cell surface protein 250 Endoplasmic CYP4A11 252 LAK*QACQLAHDHTDGVIK 271 reticulum or golgi 251 Enzyme, misc. CYP51A1 436 YLQDNPASGEK*FAYVPFGAGR 272 252 Enzyme, misc. CYP51A1 436 LDFNPDRYLQDNPASGEK*FAYVP 273 FGAGR 253 Endoplasmic Cyp7a1 127 SIDPSDGNTTENINK*TFNK 274 reticulum or golgi 254 Enzyme, misc. CYP8B1 366 VVQEDYVLK*MASGQEYQIR 275 255 Lipid binding DBI 50 QATVGDVNTDRPGLLDLK*GK 276 protein 256 Adhesion or desmoplakin 152 QMGQPCDAYQK*R 277 extracellular matrix protein 257 Adhesion or desmoplakin 166 ALYK*AISVPR 278 extracellular matrix protein 258 Adhesion or desmoplakin 249 WQLDK*IK 279 extracellular matrix protein 259 Enzyme, misc. Diminuto 446 VK*HFEAR 280 260 Unassigned DNAJA2 158 SGAVQK*CSACR 281 261 Enzyme, misc. DPYD 875 VAELMGQK*LPSFGPYLEQR 282 262 Adhesion or DSC2 838 LGDK*VQFCHTDDNQK 283 extracellular matrix protein

263 Receptor, DYSF 1612 ISIGK*K 284 channel, transporter or cell surface protein 264 Translation eEF-2 271 YFDPANGK*FSK 285 265 Translation eEF-2 274 FSK*SANSPDGK 286 266 Translation eIF3C 860 TEPTAQQNLALQLAEK*LGSLVENN 287 ER 267 Translation eIF3-theta 420 EQPEK*EPELQQYVPQLQNNTILR 288 268 Translation eIF3-theta 775 QSVYEEK*LKQFEER 289 269 Vesicle protein epsin 1 107 ENMYAVQTLK*DFQYVDRDGKDQ 290 GVNVR 270 Enzyme, misc. esterase D 17 CFGGLQK*VFEHSSVELK 291 271 Lipid binding FABP1 20 YQLQSQENFEPFMK*AIGLPEDLIQK 292 protein 272 Lipid binding FABP1 31 AIGLPEDLIQK*GK 293 protein 273 Lipid binding FABP1 36 GKDIK*GVSEIVHEGK 294 protein 274 Lipid binding FABP1 36 DIK*GVSEIVHEGK 295 protein 275 Lipid binding FABP1 46 GVSEIVHEGK*K 296 protein 276 Lipid binding FABP1 80 VK*AVVKLEGDNK 297 protein 277 Lipid binding FABP1 84 AVVK*LEGDNK 298 protein 278 Lipid binding FABP1 99 M#VTTFKGIK* 299 protein 279 Receptor, FADS2 28 WEEIQK*HNLR 300 channel, transporter or cell surface protein 280 Receptor, FADS2 87 FLK*PLLIGELAPEEPSLDR 301 channel, transporter or cell surface protein 281 Enzyme, misc. FAH 186 RPMGQMRPDNSK*PPVYGACR 302 282 Enzyme, misc. FBXL11 808 AKIRGSYLTVTLQRPTK* 303 283 Enzyme, misc. FDPS 293 QILEENYGQK*DPEKVAR 304 284 Enzyme, misc. FDPS 297 QILEENYGQKDPEK*VAR 305 285 Enzyme, misc. FMO3 209 VLVIGLGNSGCDIAAELSHVAQK*V 306 TISSR 286 Enzyme, misc. Fmo5 259 NNYMEK*QMNQR 307 287 Unassigned FUND2 123 SK*AEEVVSFVKKNVLVTGGFFGG 308 FLLGMAS 288 Apoptosis G6PI 226 TFTTQETITNAETAK*EWFLEAAKD 309 PSAVAK 289 Mitochondrial GAPDH; 212; 213; GAAQNIIPASTGAAK*AVGK 310 protein Gm10291; 195; 219; Gm13882; 231 EG622339; LOC638833 290 Mitochondrial GAPDH; 256; 331; LEKPAKYDDIK*K 311 protein LOC676923; 239; 259; Gm13882; 275; 476 LOC1000438 39; LOC638833; LOC675602 291 Enzyme, misc. GDA 133 TLK*NGTTTACYFGTIHTDSSLILAEI 312 TDKFGQR 292 G protein or G- 33 VSK*ASADLMSYCEEHAR 313 regulator gamma(12) 293 Enzyme, misc. GLUL 95 KDPNK*LVLCEVFK 314 294 Enzyme, misc. GNMT 96 YALK*ER 315 295 Enzyme, misc. GSTA2; 141; 141; VLK*SHGQDYLVGNR 316 GSTA5; 141 GSTA3 296 Enzyme, misc. GSTA3 64 SDGSLM#FQQVPMVEIDGM#K*LV 317 QTK 297 Enzyme, misc. GSTA3 64 SDGSLMFQQVPM#VEIDGM#K*LV 318 QTK 298 Enzyme, misc. GSTA3 64 SDGSLMFQQVPM#VEIDGMK*LVQ 319 TK 299 Enzyme, misc. GSTM1 198 ISAYMK*SSR 320 300 Enzyme, misc. GSTM1; 51; 51; 52 FK*LGLDFPNLPYLIDGSHK 321 GSTM5; GSTM4 301 Enzyme, misc. GSTM1; 68; 68; 69 LGLDFPNLPYLIDGSHK*ITQSNAILR 322 GSTM5; GSTM4 302 Enzyme, misc. GSTP1 127 ALPGHLK*PFETLLSQNQGGK 323 303 Unassigned Gstt3 218, 229 AK*DM#PPLMDPALK* 324 304 Enzyme, misc. Gulo 332 AMLEAHPK*VVAHYPVEVR 325 305 Enzyme, misc. Gulo 332 AM#LEAHPK*VVAHYPVEVR 326 306 Chromatin, H1F0 59 SHYK*VGENADSQIK 327 DNA-binding, DNA repair or DNA replication protein 307 Unassigned H2AE 126 VTIAQGGVLPNIQAVLLPKKTESHH 328 K*PK 308 Chromatin, H2AX 127 KSSATVGPK*APAVGK 329 DNA-binding, DNA repair or DNA replication protein 309 Chromatin, H2B; H2B2E; 5; 6; 6 PEPAK*SAPAPK 330 DNA-binding, H2B1C DNA repair or DNA replication protein 310 Receptor, HBA1 12 SNIK*AAWGK 331 channel, transporter or cell surface protein 311 Receptor, HBA1 17 AAWGK*IGGHGAEYGAEALER 332 channel, transporter or cell surface protein 312 Receptor, HBA1 41 M#FASFPTTK*TYFPHFDVSHGSAQ 333 channel, VK transporter or cell surface protein 313 Receptor, HBA1 41 MFASFPTTK*TYFPHFDVSHGSAQ 334 channel, VK transporter or cell surface protein 314 Receptor, HBA1 57 TYFPHFDVSHGSAQVK*GHGK 335 channel, transporter or cell surface protein 315 Receptor, HBA1 91 VADALASAAGHLDDLPGALSALSD 336 channel, LHAHK*LR transporter or cell surface protein 316 Receptor, HBA1 91 KVADALASAAGHLDDLPGALSALS 337 channel, DLHAHK*LR transporter or cell surface protein 317 Receptor, HBB 17 SAVSCLWAK*VNPDEVGGEALGR 338 channel, transporter or cell surface protein 318 Receptor, HBB 59 YFDSFGDLSSASAIMGNPK*VK 339 channel, transporter or cell surface protein 319 Receptor, HBB 82 NLDNLK*GTFASLSELHCDKLHVDP 340 channel, ENFR transporter or cell surface protein 320 Receptor, HBD 17 AAVSCLWGK*VNSDEVGGEALGR 341 channel, transporter or cell surface protein 321 Receptor, HBD 59 YFDSFGDLSSASAIM#GNAK*VK 342 channel, transporter or cell surface protein 322 Receptor, HBD 82 VITAFNDGLNHLDSLK*GTFASLSEL 343 channel, HCDKLHVDPENFR transporter or cell surface protein 323 Enzyme, misc. HGD 71 ILPSVSHK*PFESIDQGHVTHNWDE 344 VGPDPNQLR 324 Enzyme, misc. HGD 252 FQGK*LFACK 345 325 RNA hnRNP A/B 88 MFVGGLSWDTSK*K 346 processing 326 RNA hnRNP A/B 89 MFVGGLSWDTSKK* 347 processing 327 RNA hnRNP A/B 237 VAQPK*EVYQQQQYGSGGR 348 processing 328 Enzyme, misc. HPD 62 EVVSHVIK*QGK 349

329 Enzyme, misc. HPD 126 IVREPWVEQDK*FGK 350 330 Enzyme, misc. HPD 129 IVREPWVEQDKFGK*VK 351 331 Enzyme, misc. HPD 131 VK*FAVLQTYGDTTHTLVEK 352 332 Enzyme, misc. HPD 131 IVREPWVEQDKFGKVK* 353 333 Enzyme, misc. HPD 236 SIVVTNYEESIK*MPINEPAPGR 354 334 Enzyme, misc. HPD 247 K*KSQIQEYVDYNGGAGVQHIALK 355 335 Enzyme, misc. HPD 248 KK*SQIQEYVDYNGGAGVQHIALK 356 336 Enzyme, misc. HPD 269 SQIQEYVDYNGGAGVQHIALK*TED 357 IITAIR 337 Enzyme, misc. HPD 296 ERGTEFLAAPSSYYK*LLR 358 338 Enzyme, misc. HPD 368 HNHQGFGAGNFNSLFK*AFEEEQA 359 LR 339 Enzyme, misc. HRSP12 66 NLGEILK*AAGCDFNNVVK 360 340 Chaperone HSC70 108 VQVEYK*GETK 361 341 Chaperone HSC70 512 LSK*EDIER 362 342 Chaperone HSC70 524 MVQEAEK*YKAEDEK 363 343 Chaperone HSC70 524 MVQEAEK*YKAEDEKQR 364 344 Chaperone HSC70 524 M#VQEAEK*YKAEDEKQR 365 345 Chaperone HSC70 583 ILDKCNEIISWLDK*NQTAEKEEFEH 366 QQK 346 Chaperone HSC70 583 ILDKCNEIISWLDK*NQTAEKEEFEH 367 QQKELEK 347 Chaperone HSC70; 345; 348 IPK*IQK 368 HSPA2 348 Endoplasmic HSD11B1 73 SEEGLQK*VVSR 369 reticulum or golgi 349 Receptor, IFITM3 24 IK*EEYEVAEMGAPHGSASVR 370 channel, transporter or cell surface protein 350 Receptor, IFITM3 24 IK*EEYEVAEM#GAPHGSASVR 371 channel, transporter or cell surface protein 351 Kinase (non- IPPK 42, 43, 64 K*K*TSEEILQHLQNIVDFGKNVMK* 372 protein) 352 G protein or IQGAP2 1024 AWVNQLETQTGEASK*LPYDVTTE 373 regulator QALTYPEVK 353 G protein or IQGAP2 1354 TPEEGK*QSQAVIEDAR 374 regulator 354 RNA IREB1 79 NIEVPFK*PAR 375 processing 355 Ubiquitin ITCH 192 VSTNGSEDPEVAASGENK*R 376 conjugating system 356 Ubiquitin ITCH 407 FIYGNQDLFATSQNKEFDPLGPLPP 377 conjugating GWEK*R system 357 Unassigned ITM2B 13 VTFNSALAQK*EAK 378 358 Unassigned JOSD1 180 GK*NCELLLVVPEEVEAHQSWR 379 359 Enzyme, misc. KHK 159 IEEHNAK*QPLPQK 380 360 Unknown KIAA1033 1089 AVAK*QQNVQSTSQDEK 381 function 361 Cytoskeletal lamin A/C 270 TYSAK*LDNAR 382 protein 362 Cytoskeletal Lamin B1 124, 134 K*ESDLSGAQIK*LR 383 protein 363 Vesicle protein LAPTM4A 224 IPEK*EPPPPYLPA 384 364 Calcium- LETM1 715 VIDLVNKEDVQISTTQVAEIVATLEK 385 binding protein *EEK 365 Receptor, LISCH 538 LLEEALK*K 386 channel, transporter or cell surface protein 366 Unassigned LOC100044494; 63, 73; 176, MQANNAK*AVSARTEAIK*ALVK 387 Gm12508 186 367 Unassigned LOC100048323 247 SYLLEK*IKEHEESLDVTNPR 388 368 Unassigned LOC100048323 343 K*HMPYTNAMVHEVQR 389 369 Unassigned LOC100048323 375 YVDLGPTSLVHEVTCDTK*FR 390 370 Unknown LOC144100 742 DQPQHLEK*ITCQQR 391 function 371 G protein or LOC435565; 406; 400; TLLK*EICLRN 392 regulator EG240327; 407 ligp1 372 Cytoskeletal MARCKS 10 TAAK*GEATAERPGEAAVASSPSK 393 protein 373 Enzyme, misc. MAT1A 54 QDPNAK*VACETVCK 394 374 Enzyme, misc. MAT1A 89 DTIK*HIGYDDSAK 395 375 Enzyme, misc. MAT1A 98 HIGYDDSAK*GFDFK 396 376 Enzyme, misc. MAT1A 352 ELLEVVNK*NFDLRPGVIVR 397 377 Enzyme, misc. MAT1A 368 DLDLK*KPIYQK 398 378 Enzyme, misc. MAT1A 374 KPIYQK*TACYGHFGR 399 379 Enzyme, misc. MAT1A 374 DLDLKKPIYQK*TACYGHFGR 400 380 Unassigned MBD2 193 SDVYYFSPSGKKFRSK* 401 381 Unassigned MCT1 467 EGKEDEASTDVDEK*PKETM#K 402 382 Unassigned MCT1 469 EGKEDEASTDVDEKPK*ETMK 403 383 Enzyme, misc. Mettl7b 241 WLPVGPHIM#GK*AVK 404 384 Enzyme, misc. Mettl7b 241 WLPVGPHIMGK*AVK 405 385 Enzyme, misc. MGST1 59 VFANPEDCAGFGKGENAK*K 406 386 Enzyme, misc. MGST1 60 VFANPEDCAGFGKGENAKK* 407 387 Transcriptional MORF4L1 117 ELQK*ANQEQYAEGK 408 regulator 388 Mitochondrial MOSC1 313 LCDPSEQALYGK*LPIFGQYFALEN 409 protein PGTIR 389 Adaptor/ MPP5 553 DYHFVSRQAFEADIAAGKFIEHGEF 410 scaffold EK*NLYGTSIDSVR 390 Receptor, MT2A 20 MDPNCSCASDGSCSCAGACK*CK 411 channel, transporter or cell surface protein 391 Mitochondrial MTX1 41 IHK*TSNPWQSPSGTLPALR 412 protein 392 Ubiquitin NEDD8 54 QMNDEK*TAADYK 413 conjugating system 393 Enzyme, misc. NGLY1 130 KVQFSQHPAAAK*LPLEQSEDPAG 414 LIR 394 Enzyme, misc. NGLY1 130 VQFSQHPAAAK*LPLEQSEDPAGLIR 415 395 Enzyme, misc. NKEF-A 109 QGGLGPMNIPLISDPK*R 416 396 Kinase (non- NME2 56 QHYIDLK*DRPFFPGLVK 417 protein) 397 Adaptor/ NOSTRIN 417 AESK*APAGGQNNPSSSPSGSTVS 418 scaffold QASK 398 Enzyme, misc. NQO2 23 SFNGSLK*K 419 399 Vesicle protein NSFL1C 127 GAK*EHGAVAVER 420 400 Receptor, NUP214 686 STQTAPSSAPSTGQK*SPRVNPPV 421 channel, PKSGSSQAKALQPPVTEK transporter or cell surface protein 401 Enzyme, misc. p67phox 354 EPKELKLSVPM#PYM#LK* 422 402 RNA PABP 1 284 KFEQMK*QDR 423 processing 403 Enzyme, misc. PAH 49 EEVGALAK*VLR 424 404 Enzyme, misc. PAH 95 SKPVLGSIIK*SLR 425 405 Enzyme, misc. PAH 149 TIQELDRFANQILSYGAELDADHPG 426 FK*DPVYR 406 Enzyme, misc. PAPSS2 174 AGEIK*GFTGIDSDYEKPETPECVLK 427 407 Kinase (non- PCK1 124 WMSEEDFEK*AFNAR 428 protein) 408 Kinase (non- PCK1 124 WM#SEEDFEK*AFNAR 429 protein) 409 Kinase (non- PCK1 471 SEATAAAEHK*GK 430 protein) 410 Cell cycle PCM-1 1089 QQNQHPEK*PR 431 regulation 411 Adaptor/ PDZK1 118 EAALNDKK*PGPGMNGAVEPCAQPR 432 scaffold 412 Phosphatase PGAM1 105 AETAAK*HGEAQVK 433 413 Vesicle protein PICALM 324 EK*QAALEEEQAR 434 414 Kinase (non- PIP5KG 97 GAIQLGIGYTVGNLSSK*PER 435 protein) 415 Protein PKG2 428 RSMSSWKLSK* 436 kinase, Ser/Thr (non- receptor) 416 Adhesion or plakophilin 2 134 AAAQYSSQK*SVEER 437 extracellular matrix protein 417 Receptor, PMP70 260 MTIMEQK*YEGEYR 438 channel, transporter or cell surface protein 418 Receptor, PMP70 576 EGGWDSVQDWMDVLSGGEK*QR 439 channel, transporter or

cell surface protein 419 Enzyme, misc. PPID; 285; 263 LQPIALSCVLNIGACKLK* 440 LOC100045251 420 Cytoskeletal profilin 1 69 SSFFVNGLTLGGQK*CSVIR 441 protein 421 Protease PSMA2 69 SVHKVEPITK*HIGLVYSGM#GPDYR 442 422 Protease PSMA6 102 ARYEAANWK*YK 443 423 Protease PSMB5 91 ATAGAYIASQTVK*K 444 424 Protease PSMC2 116 YIINVK*QFAK 445 425 Protease PSMC2 116 IINADSEDPKYIINVK*QFAK 446 426 Transcriptional PSMC3 279 DAFALAK*EK 447 regulator 427 Transcriptional PSMC3 279 DAFALAK*EKAPSIIFIDELDAIGTK 448 regulator 428 Protease PSMC6 48 SENDLK*ALQSVGQIVGEVLK 449 429 Protease PSMC6 197 AVASQLDCNFLK*VVSSSIVDK 450 430 Protease PSMC6 197 AVASQLDCNFLK*VVSSSIVDKYIGE 451 SAR 431 Protease PSMD13 115 SSDEAVILCK*TAIGALK 452 432 Protease PSMD4 122 IIAFVGSPVEDNEK*DLVK 453 433 Transcriptional PTRF 163 NFKVM#IYQDEVK* 454 regulator 434 Enzyme, misc. PYGL 169 YEYGIFNQK*IR 455 435 Enzyme, misc. PYGL 803 AWNTM#VLK*NIAASGK 456 436 Enzyme, misc. PYGL 803 AWNTMVLK*NIAASGK 457 437 G protein or Rab2 165 TASNVEEAFINTAK*EIYEK 458 regulator 438 Cytoskeletal radixin 79 KENPLQFK*FR 459 protein 439 Cytoskeletal radixin 211 IAQDLEMYGVNYFEIKNK*K 460 protein 440 G protein or RALBP1 186 KKPIQEPEVPQM#DAPSVK* 461 regulator 441 Unassigned RGN 233 LDPETGK*R 462 442 Unassigned Rhbdd3 268 LGPGQLTWK*NSER 463 443 G protein or RhoA 135 MK*QEPVKPEEGR 464 regulator 444 Unknown RNF185 105 EK*TPPRPQGQRPEPENR 465 function 445 Ubiquitin RNF20 610 DSVKDKEK*GKHDDGR 466 conjugating system 446 Ubiquitin RNF5 93 LK*TPPRPQGQRPAPESR 467 conjugating system 447 Unassigned Rnft1 382 EKTCPLCRTVISECINK* 468 448 Translation RPL12; 61; 30 ITVK*LTIQNR 469 EG633570 449 Translation RPL17 95 KSAEFLLHMLK*NAESNAELK 470 450 Unassigned RPL18 78 ENK*TAVVVGTVTDDVR 471 451 Translation RPL19 186 KEEIIK*TLSKEEETKK 472 452 Translation RPL19 190 TLSK*EEETKK 473 453 Translation RPL19 195 TLSKEEETK*K 474 454 Unassigned RPL29; 134; 134; APAK*AQASAPAQAPK 475 LOC100044494; 247; 148 Gm12508; Gm7934 455 Unassigned RPL29; 151; 151; AQASAPAQAPKGAQAPK* 476 LOC100044494; 264; 165 Gm12508; Gm7934 456 Translation RPL3 293 IGQGYLIK*DGK 477 457 Translation RPL3 299 LIK*NNASTDYDLSDK 478 458 Translation RPL4 294 ILK*SPEIQR 479 459 Translation RPL4 333 LNPYAK*TMR 480 460 Translation RPL4 364 KLEAAATALATK*SEK 481 461 Unassigned RPL9; 21; 21 TILSNQTVDIPENVEITLK*GR 482 Gm10117 462 Unassigned RPLP2 24 YVASYLLAALGGNSSPSAKDIK* 483 463 Enzyme, misc. RPN1 539 LK*TEGSDLCDRVSEMQK 484 464 Translation RPS10 138 SAVPPGADK*K 485 465 Translation RPS10 138 RSAVPPGADK*K 486 466 Translation RPS10 138 SAVPPGADK*KAEAGAGSATEFQFR 487 467 Translation RPS10 138, 139 SAVPPGADK*K*AEAGAGSATEFQFR 488 468 Translation RPS10 139 RSAVPPGADKK* 489 469 Translation RPS10 139 SAVPPGADKK*AEAGAGSATEFQFR 490 470 Translation RPS12 129 DVIEEYFK*CKK 491 471 Translation RPS17 18 VIIEK*YYTR 492 472 Translation RPS2; 176; 158; IGK*PHTVPCK 493 Gm8841; 67; 171 EG625055; Gm5978 473 Translation RPS2; 58; 58; 54 AEDK*EWIPVTK 494 Gm8841; Gm5978 474 Unassigned RPS20 34 SLEK*VCADLIR 495 475 Translation RPS21 51 FNGQFK*TYGICGAIR 496 476 Translation RPS25 114 NTK*GGDAPAAGEDA 497 477 Translation RPS3 214 KPLPDHVSIVEPK*DEILPTTPISEQK 498 478 Translation RPS3 214 IGPKKPLPDHVSIVEPK*DEILPTTPI 499 SEQK 479 Translation RPS3 230 GGK*PEPPAMPQPVPTA 500 480 Translation RPS3a 45 NIGK*TLVTR 501 481 Translation RPS7 10 IVK*PNGEKPDEFESGISQALLELE 502 M#NSDLK 482 Translation RPS7 15 IVKPNGEK*PDEFESGISQALLELE 503 M#NSDLK 483 Translation RPS7 15 IVKPNGEK*PDEFESGISQALLELE 504 MNSDLK 484 Translation RRBP1 145 K*VAKVEPAVSSIVNSIQVLASK 505 485 Translation RRBP1 166 VEPAVSSIVNSIQVLASK*SAILEATPK 506 486 Translation RRBP1 219, 249, KGEGAQNQGK*KGEGAQNQAK 507 289 487 Translation RRBP1 229, 299, KGEGAQNQAK*KGEGAQNQAK 508 359, 500 488 Translation RRBP1 259, 339, KGEGAQNQAK*KGEGGQNQAK 509 480 489 Translation RRBP1 269 KGEGGQNQAK*KGEGAQNQGK 510 490 Translation RRBP1 279, 601, KGEGAQNQGK*KGEGAQNQGK 511 611, 621, 631, 641, 651, 681, 691 491 Translation RRBP1 369, 510 KGEGAQNQAK*KGEGVQNQAK 512 492 Translation RRBP1 440, 581 IEGAQNQGK*KPEGTSNQGK 513 493 Translation RRBP1 440, 581 KIEGAQNQGK*KPEGTSNQGK 514 494 Translation RRBP1 671 KGEGPQNQAK*KGEGAQNQGK 515 495 Translation RRBP1 752 TDTVANQGTK*QEGVSNQVK 516 496 Translation RRBP1 752 KTDTVANQGTK*QEGVSNQVK 517 497 Translation RRBP1 823 ASM#VQSQEAPK*QDAPAK 518 498 Translation RRBP1 823 ASMVQSQEAPK*QDAPAK 519 499 Enzyme, misc. SAHH 46 EMYSASKPLK*GAR 520 500 Enzyme, misc. SAHH 166 GISEETTTGVHNLYK*M#MSNGILK 521 501 Enzyme, misc. SAHH 166 GISEETTTGVHNLYK*MMSNGILK 522 502 Enzyme, misc. SAHH 166 GISEETTTGVHNLYK*MM#SNGILK 523 503 Enzyme, misc. SAHH 166 GISEETTTGVHNLYK*M#MSNGILK 524 VPAINVNDSVTK 504 Enzyme, misc. SAHH 166 GISEETTTGVHNLYK*MMSNGILKV 525 PAINVNDSVTK 505 Enzyme, misc. SAHH 166 GISEETTTGVHNLYK*M#M#SNGIL 526 KVPAINVNDSVTK 506 Enzyme, misc. SAHH 174 GISEETTTGVHNLYKM#MSNGILK* 527 507 Enzyme, misc. SAHH 174 GISEETTTGVHNLYKMM#SNGILK* 528 VPAINVNDSVTK 508 Enzyme, misc. SAHH 186 VPAINVNDSVTK*SK 529 509 Enzyme, misc. SAHH 188 SK*FDNLYGCR 530 510 Adaptor/ SAKS1 105 MLELVAQK*QR 531 scaffold 511 Lipid binding SEC14L2 11 VGDLSPK*QEEALAK 532 protein 512 Lipid binding SEC14L2 275 DQVK*QQYEHTVQVSR 533 protein 513 Vesicle protein SEC31L1 791 AQGK*PVSGQESSQSPYER 534 514 Unassigned SELENBP1; 342; 342 QYDISNPQK*PR 535 SELENBP2 515 Protein SgK307 1148, 1153 NTSLTDIQDLSSITYDQDGYFK*ETS 536 kinase, YK*TPKLK Ser/Thr (non- receptor) 516 Chaperone SGTA 161 AIGIDPGYSK*AYGR 537 517 Unassigned SLC22A1 319 KVPPADLK*MMCLEEDASER 538

518 Receptor, SLC26A1 32 RQPPVSQGLLETLK*AR 539 channel, transporter or cell surface protein 519 Endoplasmic SLC27A5 163 LK*DAVIQNTR 540 reticulum or golgi 520 Endoplasmic SLC27A5 599 VGMAAVK*LAPGK 541 reticulum or golgi 521 Endoplasmic SLC27A5 667 EGFDVGIIADPLYILDNK*AQTFR 542 reticulum or golgi 522 Unassigned Slc38a3 45 TEDTQHCGEGK*GFLQK 543 523 Unassigned Slc38a3 50 GFLQK*SPSKEPHFTDFEGK 544 524 Unassigned Slc38a3 54 SPSK*EPHFTDFEGK 545 525 Unassigned Slc40a1 240 AALK*VEESELK 546 526 Receptor, SLCO1A1 647 LTEK*ESECTDVCR 547 channel, transporter or cell surface protein 527 Receptor, SLCO1B3 683 KFTDEGNPEPVNNNGYSCVPSDE 548 channel, K*NSETPL transporter or cell surface protein 528 Unassigned SLCO2A1 61 SSLTTIEK* 549 529 Unassigned SLCO2B1 676 TTVK*SSELQQL 550 530 Apoptosis SOD1 136 QDDLGKGGNEESTK*TGNAGSR 551 531 Endoplasmic SRP68 38 SAGGDENK*ENERPSAGSK 552 reticulum or golgi 532 Adaptor/ ST13 355 YQSNPK*VMNLISK 553 scaffold 533 Receptor, STEAP4 97 EHYDSLTELVDYLK*GK 554 channel, transporter or cell surface protein 534 Enzyme, misc. SULT1A1 93 IPFLEFSCPGVPPGLETLK*ETPAPR 555 535 Enzyme, misc. SULT2A1 90 SPWIETDIGYSALINK*EGPR 556 536 Unassigned SYNC1 37 M#ASPEPLRGGDGARASREPHTE 557 ASFPLQESESPKEAK* 537 Adaptor/ SYNE2 5243 QSSLTM#DGGDVPLLEDMASGIVE 558 scaffold LFQK*K 538 Enzyme, misc. TALDO1 258 ALAGCDFLTISPK*LLGELLK 559 539 Enzyme, misc. TALDO1 265 LLGELLK*DNSK 560 540 Enzyme, misc. TALDO1 277 LAPALSVK*AAQTSDSEKIHLDEK 561 541 Protein Titin 855 ELSATSSTQK*ITK 562 kinase, Ser/Thr (non- receptor) 542 Enzyme, misc. TKT 260 GITGIEDKEAWHGK*PLPK 563 543 Enzyme, misc. TKT 281 NMAEQIIQEIYSQVQSK*K 564 544 Unassigned TMEM59 315 SQTEEHEEAGPLPTK*VNLAHSEI 565 545 Vesicle protein TOLLIP 143 IAWTHITIPESLK*QGQVEDEWYSL 566 SGR 546 Unassigned TRPM8 283, 298 NQLEK*YISERTSQDSNYGGK*IPIV 567 CFAQGGGRETLK 547 Cell cycle TSGA2 35 NEVGERHGHGK*AR 568 regulation 548 Cytoskeletal TUBB2C; 216; 216; TLK*LTTPTYGDLNHLVSATMSGVT 569 protein TUBB; 216; 216; TCLR TUBB2A; 216 TUBB2B; TUBB4 549 Enzyme, misc. TXNL1 180 LYSMK*FQGPDNGQGPK 570 550 Ubiquitin UBE1 604 KPLLESGTLGTK*GNVQVVIPFLTE 571 conjugating SYSSSQDPPEK system 551 Ubiquitin UBE1 627 GNVQVVIPFLTESYSSSQDPPEK*S 572 conjugating IPICTLK system 552 Ubiquitin UBE1 635 SIPICTLK*NFPNAIEHTLQWAR 573 conjugating system 553 Ubiquitin Ube1y1; 184; 185 GIK*LVVADTR 574 conjugating UBE1 system 554 Ubiquitin UBE2D3; 128; 128 IYK*TDRDKYNR 575 conjugating UBE2D4 system 555 Chromatin, UBE2N 82 IYHPNVDK*LGR 576 DNA-binding, DNA repair or DNA replication protein 556 Chromatin, UBE2N 92 ICLDILK*DKWSPALQIR 577 DNA-binding, DNA repair or DNA replication protein 557 Chromatin, UBE2N 94 ICLDILKDK*WSPALQIR 578 DNA-binding, DNA repair or DNA replication protein 558 Ubiquitin UBE2Q1 216, 232, K*SEDDGIGKENLAILEK*IK* 579 conjugating 234 system 559 Ubiquitin ubiquitin; 113; 113 VDENGK*ISR 580 conjugating LOC388720 system 560 Ubiquitin ubiquitin; 113; 113 YYKVDENGK*ISR 581 conjugating LOC388720 system 561 Ubiquitin ubiquitin; 152; 152 CCLTYCFNK*PEDK 582 conjugating LOC388720 system 562 Ubiquitin UBQLN1 53 EKEEFAVPENSSVQQFK*EEISKR 583 conjugating system 563 Enzyme, misc. UGP2 183 VK*IYTFNQSR 584 564 Mitochondrial uricase 118 AHVYVEEVPWK*R 585 protein 565 Mitochondrial uricase 220 DIVLQK*FAGPYDKGEYSPSVQK 586 protein 566 Protease USP33 227 SRPGSVVPANLFQGIK*TVNPTFR 587 567 Protease USP5 357 YVDK*LEKIFQNAPTDPTQDFSTQV 588 AK 568 Protease USP5 360 YVDKLEK*IFQNAPTDPTQDFSTQV 589 AK 569 Protease USP5 360 KYVDKLEK*IFQNAPTDPTQDFSTQ 590 VAK 570 Protease USP5 575 FASFPDYLVIQIKK* 591 571 Cytoskeletal utrophin 50 SGK*PPISDM#FSDLKDGR 592 protein 572 Enzyme, misc. VARS 951 HFCNK*LWNATK 593 573 Chromatin, VCP 109 LGDVISIQPCPDVK*YGKR 594 DNA-binding, DNA repair or DNA replication protein 574 Chromatin, VCP 336 IVSQLLTLMDGLK*QR 595 DNA-binding, DNA repair or DNA replication protein 575 Chromatin, VCP 505 ELQELVQYPVEHPDKFLK* 596 DNA-binding, DNA repair or DNA replication protein 576 Chromatin, VCP 668 KSPVAK*DVDLEFLAK 597 DNA-binding, DNA repair or DNA replication protein 577 Receptor, VDAC-1 287 NVNAGGHK*LGLGLEFQA 598 channel, transporter or cell surface protein 578 RNA vigilin 494 IEGDPQGVQQAK*R 599 processing 579 Unknown WDR19 1171, 1185 IHVKSGDHMK*GARM#LIRVANNIS 600 function K* 580 Transcriptional YB-1 168 NYQQNYQNSESGEK*NEGSESAP 601 regulator EGQAQQR 581 Transcriptional ZNF318 1246, 1250, EVK*EDDK*APGELEEQLSEDGSAP 602 regulator 1268 EK*GEVKGNASLR %in Ubiquitinated Residue Number indicates ubiquitination sites described in scientific literature K* in Peptide Sequence indicates lysine residues modified with Gly-Gly from ubiquitin or ubiquitin-like proteins, i.e., Lys-epsilon-Gly-Gly

[0129] This experiment resulted in the identification of 581 peptides that had been modified by ubiquitin or ubiquitin-like protein and allowed for the localization of the specific sites of ubiquitination within the predicate polypeptide. The experiment identified 6 of 7 known ubiquitination sites in ubiquitin itself. (See rows 39-48 of Table 4; Ikeda F, Dikic I. Atypical ubiquitin chains: new molecular signals. `Protein Modifications: Beyond the Usual Suspects` review series. EMBO Rep. 2008 June; 9(6):536-42.

[0130] Additionally, novel ubiquitination sites in enzymes responsible for linking ubiquitin to other proteins as part of the ubiquitin conjugating system were discovered (see rows 550-562 in Table 4).

[0131] Neddylation sites in ubiquitin-like molecules such as NEDD8 (see row 392 in Table 4) were also identified as trypsin digestion of neddylated proteins leaves the same K(GG) remnant as trypsin digestion of ubiquitinated proteins. Thus, the invention contemplates the use of the antibodies described herein in, for example, the methods described herein to identify neddylated proteins following digestion of such neddylated proteins with a hydrolyzing agent such as trypsin. NEDD8 is about 60% identical to ubiquitin and like ubiquitin can form polyneddylation chains (Jones J, Wu K, Yang Y, Guerrero C, Nillegoda N, Pan Z Q, Huang L. A targeted proteomic analysis of the ubiquitin-like modifier nedd8 and associated proteins. J Proteome Res. 2008 March; 7(3):1274-87). Several known ubiquitination sites in histones (e.g., H2A and H2B; see rows 13-26 and 27-30, respectfully, in Table 4) were identified. Ubiquitination of these histones is thought to regulate many nuclear processes such as transcription, silencing, and DNA repair (Weake V M, Workman J L. Histone ubiquitination: triggering gene activity. Mol Cell. 2008 Mar. 28; 29(6):653-63).

[0132] The invention also contemplates the use of the antibodies described herein in, for example, the methods described herein to identify proteins modified by the Interferon-induced 17 kDa protein, also called the ISG15 protein because it is encoded by the ISG15 gene (see Blomstrom et al., J Biol Chem 261 (19): 8811-8816, 1986). Following digestion of such ISG15-modified proteins with a hydrolyzing agent such as trypsin, the antibodies of the invention will specifically bind to and recognize the modified lysine residues in the hydrolyzed ISG15-modified proteins (see, e.g., Zhao et al., Proc. Natl. Acad. Sci 107(5): 2253-2258, 2010).

[0133] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of this invention. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used to practice this invention, the preferred compositions, methods, kits, and means for communicating information are described herein.

[0134] All references cited above are incorporated herein by reference in their entirety to the extent allowed by law. The discussion of those references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference.

Sequence CWU 1

6021471PRTArtificial SequenceSynthetic Peptide 1Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly1 5 10 15Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro 20 25 30Gly Thr Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Thr Ile Ser 35 40 45Ser Asn Tyr Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Ile Ala Cys Ile Tyr Gly Gly Ser Ser Gly Thr Thr Leu Tyr65 70 75 80Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr 85 90 95Thr Val Thr Leu Lys Met Pro Ser Leu Thr Ala Ala Asp Thr Ala Thr 100 105 110Tyr Phe Cys Ala Arg Asp Phe Arg Gly Ala Asp Tyr Ser Ser Tyr Asp 115 120 125Arg Ile Trp Asp Thr Arg Leu Asp Leu Trp Gly Gln Gly Thr Leu Val 130 135 140Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala145 150 155 160Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu 165 170 175Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly 180 185 190Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser 195 200 205Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln 210 215 220Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp225 230 235 240Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro 245 250 255Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys 260 265 270Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn 290 295 300Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe305 310 315 320Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Ala His Gln Asp 325 330 335Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu 340 345 350Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu 355 360 365Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser 370 375 380Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp385 390 395 400Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys 405 410 415Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser 420 425 430Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr 435 440 445Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460Ile Ser Arg Ser Pro Gly Lys465 4702238PRTArtificial SequenceSynthetic Peptide 2Met Asp Met Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Leu Pro Gly Val Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30Val Ser Ala Ala Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ser Ser 35 40 45Glu Asn Val Tyr Asn Lys Asn Trp Leu Ser Trp Tyr Gln Gln Lys Pro 50 55 60Gly Gln Pro Pro Lys Leu Leu Ile Gln Lys Ala Ser Thr Leu Ala Ser65 70 75 80Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95Leu Thr Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys 100 105 110Ala Gly Asp Tyr Gly Gly Thr Gly Asp Ala Phe Val Phe Gly Gly Gly 115 120 125Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile 130 135 140Phe Pro Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val145 150 155 160Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val 165 170 175Asp Gly Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln 180 185 190Asn Ser Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr 195 200 205Ser Thr Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln 210 215 220Gly Thr Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys225 230 235313PRTArtificial SequenceSynthetic Peptide 3Gly Phe Thr Ile Ser Ser Asn Tyr Tyr Ile Tyr Trp Val1 5 10418PRTArtificial SequenceSynthetic Peptide 4Cys Ile Tyr Gly Gly Ser Ser Gly Thr Thr Leu Tyr Ala Ser Trp Ala1 5 10 15Lys Gly520PRTArtificial SequenceSynthetic Peptide 5Asp Phe Arg Gly Ala Asp Tyr Ser Ser Tyr Asp Arg Ile Trp Asp Thr1 5 10 15Arg Leu Asp Leu 20620PRTArtificial SequenceSynthetic Peptide 6Asp Phe Arg Gly Ala Asp Tyr Ser Ser Tyr Asp Arg Ile Trp Asp Thr1 5 10 15Arg Leu Asp Leu 2077PRTArtificial SequenceSynthetic Peptide 7Lys Ala Ser Thr Leu Ala Ser1 5812PRTArtificial SequenceSynthetic Peptide 8Ala Gly Asp Tyr Gly Gly Thr Gly Asp Ala Phe Val1 5 10911PRTArtificial SequenceSynthetic Peptide 9Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys1 5 101021PRTArtificial SequenceSynthetic Peptide 10Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr1 5 10 15Ile Glu Asn Val Lys 201123PRTArtificial SequenceSynthetic Peptide 11Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr1 5 10 15Ile Glu Asn Val Lys Ala Lys 201231PRTArtificial SequenceSynthetic Peptide 12Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys1 5 10 15Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg 20 25 301315PRTArtificial SequenceSynthetic Peptide 13Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg1 5 10 151415PRTArtificial SequenceSynthetic Peptide 14Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg1 5 10 151513PRTArtificial SequenceSynthetic Peptide 15Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg1 5 101615PRTArtificial SequenceSynthetic Peptide 16Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg1 5 10 151715PRTArtificial SequenceSynthetic Peptide 17Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg1 5 10 151812PRTArtificial SequenceSynthetic Peptide 18Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg1 5 101921PRTArtificial SequenceSynthetic Peptide 19Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp1 5 10 15Tyr Asn Ile Gln Lys 202030PRTArtificial SequenceSynthetic Peptide 20Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp1 5 10 15Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg 20 25 302118PRTArtificial SequenceSynthetic Peptide 21Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val1 5 10 15Leu Arg2214PRTArtificial SequenceSynthetic Peptide 22Met Ser Leu Lys Gly Thr Thr Val Thr Pro Asp Lys Arg Lys1 5 102313PRTArtificial SequenceSynthetic Peptide 23Met Ser Leu Lys Gly Thr Thr Val Thr Pro Asp Lys Arg1 5 102412PRTArtificial SequenceSynthetic Peptide 24Met Ser Leu Lys Gly Thr Thr Val Thr Pro Asp Lys1 5 102513PRTArtificial SequenceSynthetic Peptide 25Met Ser Leu Lys Gly Thr Thr Val Thr Pro Asp Lys Arg1 5 102614PRTArtificial SequenceSynthetic Peptide 26Met Ser Leu Lys Gly Thr Thr Val Thr Pro Asp Lys Arg Lys1 5 102720PRTArtificial SequenceSynthetic Peptide 27Met Gln Glu Asp Ile Glu Met Thr Lys Thr Gln Ser Ile Tyr Asp Asp1 5 10 15Lys Asn His Arg 202819PRTArtificial SequenceSynthetic Peptide 28Lys Ala Ser Gly Pro Pro Val Ser Glu Leu Ile Thr Lys Ala Val Ala1 5 10 15Ala Ser Lys2912PRTArtificial SequenceSynthetic Peptide 29Lys Ala Leu Ala Ala Ala Gly Tyr Asp Val Glu Lys1 5 103016PRTArtificial SequenceSynthetic Peptide 30Lys Ala Leu Ala Ala Ala Gly Tyr Asp Val Glu Lys Asn Asn Ser Arg1 5 10 153115PRTArtificial SequenceSynthetic Peptide 31Ala Leu Ala Ala Ala Gly Tyr Asp Val Glu Lys Asn Asn Ser Arg1 5 10 153219PRTArtificial SequenceSynthetic Peptide 32Lys Thr Ser Gly Pro Pro Val Ser Glu Leu Ile Thr Lys Ala Val Ala1 5 10 15Ala Ser Lys3318PRTArtificial SequenceSynthetic Peptide 33Thr Ser Gly Pro Pro Val Ser Glu Leu Ile Thr Lys Ala Val Ala Ala1 5 10 15Ser Lys3426PRTArtificial SequenceSynthetic Peptide 34Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile Gln Ala Val Leu1 5 10 15Leu Pro Lys Lys Thr Glu Ser His His Lys 20 253526PRTArtificial SequenceSynthetic Peptide 35Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile Gln Ala Val Leu1 5 10 15Leu Pro Lys Lys Thr Glu Ser His His Lys 20 253620PRTArtificial SequenceSynthetic Peptide 36Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile Gln Ala Val Leu1 5 10 15Leu Pro Lys Lys 203720PRTArtificial SequenceSynthetic Peptide 37Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile Gln Ala Val Leu1 5 10 15Leu Pro Lys Lys 203828PRTArtificial SequenceSynthetic Peptide 38Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile Gln Ala Val Leu1 5 10 15Leu Pro Lys Lys Thr Glu Ser His His Lys Pro Lys 20 253925PRTArtificial SequenceSynthetic Peptide 39Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile Gln Ala Val Leu1 5 10 15Leu Pro Lys Lys Thr Glu Ser Gln Lys 20 254025PRTArtificial SequenceSynthetic Peptide 40Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile Gln Ala Val Leu1 5 10 15Leu Pro Lys Lys Thr Glu Ser Gln Lys 20 254121PRTArtificial SequenceSynthetic Peptide 41Gly Val Thr Ile Ala Ser Gly Gly Val Leu Pro Asn Ile His Pro Glu1 5 10 15Leu Leu Ala Lys Lys 204222PRTArtificial SequenceSynthetic Peptide 42Gly Val Thr Ile Ala Ser Gly Gly Val Leu Pro Asn Ile His Pro Glu1 5 10 15Leu Leu Ala Lys Lys Arg 204322PRTArtificial SequenceSynthetic Peptide 43Gly Val Thr Ile Ala Ser Gly Gly Val Leu Pro Asn Ile His Pro Glu1 5 10 15Leu Leu Ala Lys Lys Arg 204426PRTArtificial SequenceSynthetic Peptide 44Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile Gln Ala Val Leu1 5 10 15Leu Pro Lys Lys Thr Glu Thr His His Lys 20 25459PRTArtificial SequenceSynthetic Peptide 45Lys Ser Ser Ala Thr Val Gly Pro Lys1 54624PRTArtificial SequenceSynthetic Peptide 46Leu Leu Gly Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile1 5 10 15Gln Ala Val Leu Leu Pro Lys Lys 204731PRTArtificial SequenceSynthetic Peptide 47Asn Asp Glu Glu Leu Asn Lys Leu Leu Gly Gly Val Thr Ile Ala Gln1 5 10 15Gly Gly Val Leu Pro Asn Ile Gln Ala Val Leu Leu Pro Lys Lys 20 25 30489PRTArtificial SequenceSynthetic Peptide 48Ala Val Thr Lys Tyr Thr Ser Ser Lys1 54914PRTArtificial SequenceSynthetic Peptide 49Val Leu Lys Gln Val His Pro Asp Thr Gly Ile Ser Ser Lys1 5 105012PRTArtificial SequenceSynthetic Peptide 50His Ala Val Ser Glu Gly Thr Lys Ala Val Thr Lys1 5 10519PRTArtificial SequenceSynthetic Peptide 51Ala Val Thr Lys Tyr Thr Ser Ala Lys1 55225PRTArtificial SequenceSynthetic Peptide 52Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile Gln Ala Val Leu1 5 10 15Leu Pro Lys Lys Thr Glu Ser His Lys 20 255325PRTArtificial SequenceSynthetic Peptide 53Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile Gln Ala Val Leu1 5 10 15Leu Pro Lys Lys Thr Glu Ser His Lys 20 25549PRTArtificial SequenceSynthetic Peptide 54Ile Thr Ile Thr Asn Asp Lys Gly Arg1 55512PRTArtificial SequenceSynthetic Peptide 55Leu Ile Tyr Ser Gly Lys Gln Met Asn Asp Glu Lys1 5 105615PRTArtificial SequenceSynthetic Peptide 56Asp Thr Gly Lys Thr Pro Val Glu Pro Glu Val Ala Ile His Arg1 5 10 155717PRTArtificial SequenceSynthetic Peptide 57Ser Ser His Pro Ser Glu Pro Pro Lys Glu Ala Ser Gly Thr Asp Val1 5 10 15Arg5830PRTArtificial SequenceSynthetic Peptide 58Glu Ala Phe Ser Ser Val Ile Ile Lys Glu Pro Gln Ile Glu Pro Thr1 5 10 15Ala Asp Leu Thr Gly Ile Thr Asn Gln Leu Ile Thr Cys Lys 20 25 305921PRTArtificial SequenceSynthetic Peptide 59Val Gly Ile Asn Tyr Gln Pro Pro Thr Val Val Pro Gly Gly Asp Leu1 5 10 15Ala Lys Val Gln Arg 206021PRTArtificial SequenceSynthetic Peptide 60Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr1 5 10 15Ile Glu Asn Val Lys 206111PRTArtificial SequenceSynthetic Peptide 61Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys1 5 106215PRTArtificial SequenceSynthetic Peptide 62Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg1 5 10 156315PRTArtificial SequenceSynthetic Peptide 63Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg1 5 10 156413PRTArtificial SequenceSynthetic Peptide 64Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg1 5 106515PRTArtificial SequenceSynthetic Peptide 65Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg1 5 10 156612PRTArtificial SequenceSynthetic Peptide 66Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg1 5 106721PRTArtificial SequenceSynthetic Peptide 67Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp1 5 10 15Tyr Asn Ile Gln Lys 206830PRTArtificial SequenceSynthetic Peptide 68Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp1 5 10 15Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg 20 25 306918PRTArtificial SequenceSynthetic Peptide 69Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val1 5 10 15Leu Arg7023PRTArtificial SequenceSynthetic Peptide 70Cys Pro Gly Leu Leu Thr Pro Ser Gln Ile Lys Pro Gly Thr Tyr Lys1 5 10 15Leu Phe Phe Asp Thr Glu Arg 207119PRTArtificial SequenceSynthetic Peptide 71Ser Met Leu Glu Ala His Gln Ala Lys His Val Lys Gln Leu Leu Ser1 5 10 15Lys Pro Arg7214PRTArtificial SequenceSynthetic Peptide 72Asn Leu Leu Ser Val Ala Tyr Lys Asn Val Val Gly Ala Arg1 5 107322PRTArtificial SequenceSynthetic Peptide 73Lys Gly Glu Ser Val Met Val Val Pro Thr Leu Ser Glu Glu Glu Ala1 5 10 15Lys Gln Cys Phe Pro Lys 207422PRTArtificial SequenceSynthetic Peptide 74Lys Gly Glu Ser

Val Met Val Val Pro Thr Leu Ser Glu Glu Glu Ala1 5 10 15Lys Gln Cys Phe Pro Lys 207511PRTArtificial SequenceSynthetic Peptide 75Gly Val Phe Thr Lys Glu Leu Pro Ser Gly Lys1 5 107614PRTArtificial SequenceSynthetic Peptide 76Thr Val Val Gly Lys Glu Glu Glu Gly Ser Asp Pro Glu Lys1 5 107717PRTArtificial SequenceSynthetic Peptide 77Thr Val Val Gly Lys Glu Glu Glu Gly Ser Asp Pro Glu Lys Ala Leu1 5 10 15Arg7812PRTArtificial SequenceSynthetic Peptide 78Ser Glu Gly Lys Gln Asp Ala Leu Glu Glu Phe Lys1 5 107918PRTArtificial SequenceSynthetic Peptide 79Glu Ile Leu Glu Lys Ala Gly Gln Ile Thr Asn Glu Ala Leu Ser Asn1 5 10 15Ile Arg8028PRTArtificial SequenceSynthetic Peptide 80Met Leu Thr Gly Phe Ala Ser Gln Asp Lys Glu Ile Leu Glu Lys Ala1 5 10 15Gly Gln Ile Thr Asn Glu Ala Leu Ser Asn Ile Arg 20 258128PRTArtificial SequenceSynthetic Peptide 81Met Leu Thr Gly Phe Ala Ser Gln Asp Lys Glu Ile Leu Glu Lys Ala1 5 10 15Gly Gln Ile Thr Asn Glu Ala Leu Ser Asn Ile Arg 20 258210PRTArtificial SequenceSynthetic Peptide 82Lys Ile Gln Val Gln Asn Met Lys Asn Lys1 5 10839PRTArtificial SequenceSynthetic Peptide 83Ile Gln Val Gln Asn Met Lys Asn Lys1 58411PRTArtificial SequenceSynthetic Peptide 84Phe Leu Leu Lys Ser Cys Pro Leu Leu Thr Lys1 5 108510PRTArtificial SequenceSynthetic Peptide 85Gly Lys Tyr Lys Glu Glu Thr Ile Glu Lys1 5 108616PRTArtificial SequenceSynthetic Peptide 86Arg Ser Lys Ala Glu Glu Leu Gly Leu Pro Ile Leu Gly Val Leu Arg1 5 10 158728PRTArtificial SequenceSynthetic Peptide 87Ile Gln Pro Gln Gln Val Ala Val Trp Pro Thr Leu Val Asp Ile Asn1 5 10 15Ser Leu Asp Ser Leu Thr Glu Ala Tyr Lys Leu Arg 20 258810PRTArtificial SequenceSynthetic Peptide 88Val Tyr Asp Lys Val Gln Asn Glu Ala Lys1 5 108914PRTArtificial SequenceSynthetic Peptide 89Asn Gln Cys Val Lys Glu Ala Ile Leu Glu Asp Leu Gln Lys1 5 109015PRTArtificial SequenceSynthetic Peptide 90Phe Phe Gln Thr Gln Ile Lys Ser Leu Tyr Glu Ser Ile Glu Glu1 5 10 159113PRTArtificial SequenceSynthetic Peptide 91Asp Leu Thr Asp Tyr Leu Met Lys Ile Leu Thr Glu Arg1 5 109213PRTArtificial SequenceSynthetic Peptide 92Glu Ile Thr Ala Leu Ala Pro Ser Thr Met Lys Ile Lys1 5 109312PRTArtificial SequenceSynthetic Peptide 93His Val Ala Asp Glu Met Leu Lys Asp Met Lys Lys1 5 109410PRTArtificial SequenceSynthetic Peptide 94Ile Lys Met Val Ala Thr Gly Val Cys Arg1 5 109512PRTArtificial SequenceSynthetic Peptide 95Ile Cys Lys His Pro Glu Ser Asn Phe Cys Ser Arg1 5 109626PRTArtificial SequenceSynthetic Peptide 96Ile Asp Gly Ala Ser Pro Leu Asp Lys Val Cys Leu Ile Gly Cys Gly1 5 10 15Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys 20 259729PRTArtificial SequenceSynthetic Peptide 97Ile Asp Gly Ala Ser Pro Leu Asp Lys Val Cys Leu Ile Gly Cys Gly1 5 10 15Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys 20 259811PRTArtificial SequenceSynthetic Peptide 98Thr Trp Lys Gly Ala Ile Phe Gly Gly Phe Lys1 5 10999PRTArtificial SequenceSynthetic Peptide 99Leu Val Ala Asp Phe Met Ala Lys Lys1 510018PRTArtificial SequenceSynthetic Peptide 100Ala Ala Thr Phe Phe Gly Cys Ile Gly Ile Asp Lys Phe Gly Glu Ile1 5 10 15Leu Lys10110PRTArtificial SequenceSynthetic Peptide 101Glu Gln Gly Phe Glu Thr Lys Asp Ile Lys1 5 1010212PRTArtificial SequenceSynthetic Peptide 102Thr Gly Cys Thr Phe Pro Glu Lys Pro Asp Phe His1 5 1010323PRTArtificial SequenceSynthetic Peptide 103Glu Lys Gln Trp Val Asp Gln Ser Ser Pro Val Leu Leu Asp Asn Pro1 5 10 15Val Leu Gly Ser Met Ala Lys 2010419PRTArtificial SequenceSynthetic Peptide 104Tyr Ile Ser Gly Ser Ser Phe Lys Asp His Pro Asp Phe Pro Phe Trp1 5 10 15Asp Glu Tyr10522PRTArtificial SequenceSynthetic Peptide 105Val Pro Lys Asp Glu Gln Gly Ser Ala Ala Asp Ser Glu Asp Ser Pro1 5 10 15Thr Ile Glu Ala Val Arg 2010632PRTArtificial SequenceSynthetic Peptide 106Cys Val Leu Ile Phe Gly Val Pro Ser Arg Val Pro Lys Asp Glu Gln1 5 10 15Gly Ser Ala Ala Asp Ser Glu Asp Ser Pro Thr Ile Glu Ala Val Arg 20 25 3010711PRTArtificial SequenceSynthetic Peptide 107Ala Ala Leu Leu Lys His Gly Leu Gly Asn Arg1 5 1010813PRTArtificial SequenceSynthetic Peptide 108Val Gly Thr Lys Cys Cys Thr Leu Pro Glu Asp Gln Arg1 5 1010911PRTArtificial SequenceSynthetic Peptide 109Arg Lys Pro Val Leu Thr Ser Gln Leu Glu Arg1 5 1011019PRTArtificial SequenceSynthetic Peptide 110Ala Asn Asn Thr Thr Tyr Gly Leu Ala Ala Gly Leu Phe Thr Lys Asp1 5 10 15Leu Asp Lys11120PRTArtificial SequenceSynthetic Peptide 111Arg Ala Asn Asn Thr Thr Tyr Gly Leu Ala Ala Gly Leu Phe Thr Lys1 5 10 15Asp Leu Asp Lys 2011213PRTArtificial SequenceSynthetic Peptide 112Ile Phe Val Glu Glu Ser Ile Tyr Glu Glu Phe Val Lys1 5 1011319PRTArtificial SequenceSynthetic Peptide 113Ala Asn Asn Thr Thr Tyr Gly Leu Ala Ala Gly Val Phe Thr Lys Asp1 5 10 15Leu Asp Lys11411PRTArtificial SequenceSynthetic Peptide 114Thr Val Ala Met Gln Ile Ser Gln Lys Asn Ser1 5 1011513PRTArtificial SequenceSynthetic Peptide 115Leu Leu Asn Lys Leu Ala Asp Leu Met Glu Arg Asp Arg1 5 1011611PRTArtificial SequenceSynthetic Peptide 116Leu Leu Asn Lys Leu Ala Asp Leu Met Glu Arg1 5 101178PRTArtificial SequenceSynthetic Peptide 117Leu Ile Lys Glu Ala Ala Gly Lys1 511821PRTArtificial SequenceSynthetic Peptide 118Trp Gly Asn Lys Gly Phe Phe Val Gln Pro Thr Val Phe Ser Asn Val1 5 10 15Thr Asp Glu Met Arg 2011921PRTArtificial SequenceSynthetic Peptide 119Trp Gly Asn Lys Gly Phe Phe Val Gln Pro Thr Val Phe Ser Asn Val1 5 10 15Thr Asp Glu Met Arg 2012015PRTArtificial SequenceSynthetic Peptide 120Ile Ala Lys Glu Glu Ile Phe Gly Pro Val Gln Gln Ile Met Lys1 5 10 151219PRTArtificial SequenceSynthetic Peptide 121Leu Gln Ser Leu Leu Lys Gly Gln Lys1 512225PRTArtificial SequenceSynthetic Peptide 122Phe Gln Asp Glu Glu Glu Val Phe Glu Trp Asn Asn Glu Val Lys Gln1 5 10 15Gly Leu Ser Ser Ser Ile Phe Thr Lys 20 2512311PRTArtificial SequenceSynthetic Peptide 123Ile Lys Val Glu Asn Thr Glu Glu Asn Arg Arg1 5 1012420PRTArtificial SequenceSynthetic Peptide 124Gly Ile Val Val Gly Ile Lys Leu Asp Gln Gly Gly Ala Pro Leu Ala1 5 10 15Gly Thr Asn Lys 2012533PRTArtificial SequenceSynthetic Peptide 125Gly Ile Val Val Gly Ile Lys Leu Asp Gln Gly Gly Ala Pro Leu Ala1 5 10 15Gly Thr Asn Lys Glu Thr Thr Ile Gln Gly Leu Asp Gly Leu Ser Glu 20 25 30Arg12626PRTArtificial SequenceSynthetic Peptide 126Leu Asp Gln Gly Gly Ala Pro Leu Ala Gly Thr Asn Lys Glu Thr Thr1 5 10 15Ile Gln Gly Leu Asp Gly Leu Ser Glu Arg 20 251279PRTArtificial SequenceSynthetic Peptide 127Ala Thr Gln Glu Ala Phe Met Lys Arg1 51288PRTArtificial SequenceSynthetic Peptide 128Phe Ser Lys Ser Ala Asp Glu Arg1 512914PRTArtificial SequenceSynthetic Peptide 129Phe Leu Asn Lys Ser Ser Glu Asp Asp Gly Ala Ser Glu Arg1 5 1013012PRTArtificial SequenceSynthetic Peptide 130Ala Ile Asn Glu Ala Tyr Lys Glu Asp Tyr His Lys1 5 101319PRTArtificial SequenceSynthetic Peptide 131Ala Ala Ile Glu His Ile Lys Gln Lys1 513211PRTArtificial SequenceSynthetic Peptide 132Leu Gly Lys Glu Val Gln Ala Ala Gln Ala Arg1 5 1013311PRTArtificial SequenceSynthetic Peptide 133Ser Lys Met Glu Glu Gln Thr Gln Gln Ile Arg1 5 1013412PRTArtificial SequenceSynthetic Peptide 134Gly Met Lys Glu Val Leu Asp Gln Ser Gly Pro Arg1 5 1013510PRTArtificial SequenceSynthetic Peptide 135Ile Val Asn Lys Ile Pro Gln Ala Thr Arg1 5 1013611PRTArtificial SequenceSynthetic Peptide 136Gly Gly Val Glu Lys Gly Pro Ala Ala Leu Arg1 5 1013714PRTArtificial SequenceSynthetic Peptide 137Tyr Phe Ser Met Thr Glu Val Asp Lys Leu Gly Ile Gly Lys1 5 1013817PRTArtificial SequenceSynthetic Peptide 138Gln Phe Cys Phe Asn Cys Gly Glu Asn Trp His Asp Pro Val Lys Cys1 5 10 15Lys13915PRTArtificial SequenceSynthetic Peptide 139His Leu Trp Asn Val Asp Val Gln Gly Ser Lys Ala Tyr Ser Arg1 5 10 1514013PRTArtificial SequenceSynthetic Peptide 140Glu Gly Ala Lys Tyr Val Ser His Gly Ala Thr Gly Lys1 5 1014115PRTArtificial SequenceSynthetic Peptide 141Tyr Val Ser His Gly Ala Thr Gly Lys Gly Asn Asp Gln Val Arg1 5 10 1514213PRTArtificial SequenceSynthetic Peptide 142His Cys Ile Gln Lys Ser Gln Glu Arg Val Glu Gly Lys1 5 101439PRTArtificial SequenceSynthetic Peptide 143His Cys Ile Gln Lys Ser Gln Glu Arg1 514427PRTArtificial SequenceSynthetic Peptide 144Lys Val Glu Leu Trp Lys Glu Gln Phe Phe Glu Asn Tyr Tyr Gly Gln1 5 10 15Ser Ser Gly Leu Ser Leu Glu Asp Ser Gln Lys 20 2514515PRTArtificial SequenceSynthetic Peptide 145Val Gln Gln Leu Val Ala Lys Glu Leu Gly Gln Ile Gly Thr Arg1 5 10 1514615PRTArtificial SequenceSynthetic Peptide 146Glu His Ile Ala Ala Ser Val Ser Ile Pro Ser Glu Lys Gln Arg1 5 10 1514715PRTArtificial SequenceSynthetic Peptide 147Thr His Gly Arg Ala Lys Ser Tyr Lys Cys Gly Glu Cys Gly Lys1 5 10 1514814PRTArtificial SequenceSynthetic Peptide 148Leu Ile Val Asn Lys Asn Ala Gly Glu Thr Leu Leu Gln Arg1 5 101498PRTArtificial SequenceSynthetic Peptide 149Trp Asp Ile Gln Lys Tyr Ala Arg1 51509PRTArtificial SequenceSynthetic Peptide 150Leu Val Ala Ser Lys Thr Asp Gly Lys1 515123PRTArtificial SequenceSynthetic Peptide 151Gly Ser Ser Leu Pro Gly Lys Pro Ser Ser Pro His Ser Gly Gln Asp1 5 10 15Pro Pro Ala Pro Pro Val Asp 2015221PRTArtificial SequenceSynthetic Peptide 152Asp Ile Lys His Asp Pro Ser Leu Gln Pro Trp Ser Ala Ser Tyr Asp1 5 10 15Pro Gly Ser Ala Lys 2015328PRTArtificial SequenceSynthetic Peptide 153Asp Ile Lys His Asp Pro Ser Leu Gln Pro Trp Ser Ala Ser Tyr Asp1 5 10 15Pro Gly Ser Ala Lys Thr Ile Leu Asn Asn Gly Lys 20 2515415PRTArtificial SequenceSynthetic Peptide 154Thr Asp Gln Gly Ile Lys Asn Leu Pro Val Gly Glu Ala Gly Arg1 5 10 1515510PRTArtificial SequenceSynthetic Peptide 155Phe Leu Ser Asp Lys Trp Met Leu Gln Lys1 5 101568PRTArtificial SequenceSynthetic Peptide 156Leu Ala Cys Lys Glu Ala Val Arg1 515718PRTArtificial SequenceSynthetic Peptide 157Asp Asp Lys His Gly Ser Tyr Glu Asn Ala Val His Ser Gly Ala Leu1 5 10 15Asp Asp15828PRTArtificial SequenceSynthetic Peptide 158Val Asp Gln Ile Ile Met Ala Lys Pro Ala Gly Gly Pro Lys Pro Pro1 5 10 15Ser Gly Lys Lys Asp Trp Asp Asp Asp Gln Asn Asp 20 2515920PRTArtificial SequenceSynthetic Peptide 159Pro Ala Gly Gly Pro Lys Pro Pro Ser Gly Lys Lys Asp Trp Asp Asp1 5 10 15Asp Gln Asn Asp 2016028PRTArtificial SequenceSynthetic Peptide 160Val Asp Gln Ile Ile Met Ala Lys Pro Ala Gly Gly Pro Lys Pro Pro1 5 10 15Ser Gly Lys Lys Asp Trp Asp Asp Asp Gln Asn Asp 20 2516112PRTArtificial SequenceSynthetic Peptide 161Ser Ile Gln Lys Leu Leu Glu Trp Glu Asn Asn Arg1 5 1016213PRTArtificial SequenceSynthetic Peptide 162Arg Thr Thr His Gly Phe Lys Met Ser Lys Ile Tyr Lys1 5 1016318PRTArtificial SequenceSynthetic Peptide 163Ser Thr Gly Pro Gly Thr Gly Thr Gly Thr Ala Tyr Asp Arg Lys Asp1 5 10 15Tyr Val1649PRTArtificial SequenceSynthetic Peptide 164Leu His Ile Val Lys Val Val Ala Lys1 516515PRTArtificial SequenceSynthetic Peptide 165Ala His Ile Ala Gln Leu Cys Glu Lys Ala Gly Leu Leu Gln Arg1 5 10 151669PRTArtificial SequenceSynthetic Peptide 166Ala Val Asn Tyr Phe Ser Lys Val Lys1 516712PRTArtificial SequenceSynthetic Peptide 167Val Lys Gln Leu Pro Leu Val Lys Pro Tyr Leu Arg1 5 1016823PRTArtificial SequenceSynthetic Peptide 168Glu Pro Leu Phe Gly Ile Ser Thr Gly Asn Ile Ile Thr Gly Leu Ala1 5 10 15Ala Gly Ala Lys Ser Tyr Lys 201699PRTArtificial SequenceSynthetic Peptide 169Ser Tyr Lys Met Ser Met Ala Asn Arg1 517028PRTArtificial SequenceSynthetic Peptide 170Gln Leu Phe Ser Asp Lys Leu Asn Glu Ile Asn Glu Lys Ile Ala Pro1 5 10 15Ser Phe Ala Val Glu Ser Met Glu Asp Ala Leu Lys 20 2517118PRTArtificial SequenceSynthetic Peptide 171Thr Ser Ala Cys Phe Glu Pro Ser Leu Asp Tyr Met Val Thr Lys Ile1 5 10 15Pro Arg17218PRTArtificial SequenceSynthetic Peptide 172Ser Ile Phe Ser Ala Val Leu Asp Glu Leu Lys Val Ala Gln Ala Pro1 5 10 15Trp Lys17312PRTArtificial SequenceSynthetic Peptide 173Glu Val Glu Met Asp Ala Val Gly Lys Glu Gly Arg1 5 1017419PRTArtificial SequenceSynthetic Peptide 174Ser Phe Pro Phe Val Ser Lys Thr Leu Gly Val Asp Phe Ile Asp Val1 5 10 15Ala Thr Lys17516PRTArtificial SequenceSynthetic Peptide 175Tyr Leu Glu Ser Val Arg Pro Leu Met Lys Glu Gly Asp Phe Gln Arg1 5 10 1517615PRTArtificial SequenceSynthetic Peptide 176Val Leu Ala Ala Cys Leu Thr Glu Lys Gly Ala Glu Gln Leu Arg1 5 10 1517730PRTArtificial SequenceSynthetic Peptide 177Leu Ser His Ser Ile Glu Lys Leu Trp Asp Gln Thr Ser Ser Glu Val1 5 10 15Lys Glu Val Tyr Asp Lys Asn Phe Leu Asp Ser Tyr Ile Lys 20 25 3017827PRTArtificial SequenceSynthetic Peptide 178Ala Val Val Leu Pro Ile Ser Leu Ala Thr Thr Phe Lys Gln Asp Phe1 5 10 15Pro Gly Gln Ser Ser Gly Phe Glu Tyr Ser Arg 20 2517914PRTArtificial SequenceSynthetic Peptide 179Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly Ala Lys1 5 1018011PRTArtificial SequenceSynthetic Peptide 180Met Ser Glu Asp Lys Pro Gln Asp Tyr Lys Lys1 5 1018111PRTArtificial SequenceSynthetic Peptide 181Met Ser Glu Asp Lys Pro Gln Asp Tyr Lys Lys1 5 1018233PRTArtificial SequenceSynthetic Peptide 182Met Glu Ile Val Asp His Ala Leu His Ala Leu Thr Asp Glu Val Ile1 5 10 15Ile Pro His Ser Gly Trp Glu Arg Glu Pro Asn Glu Asp Cys Lys Pro 20 25 30Arg18333PRTArtificial SequenceSynthetic Peptide 183Met Glu Ile Val Asp His Ala Leu His Ala Leu Thr Asp Glu Val Ile1 5

10 15Ile Pro His Ser Gly Trp Glu Arg Glu Pro Asn Glu Asp Cys Lys Pro 20 25 30Arg18422PRTArtificial SequenceSynthetic Peptide 184Ser Ala Leu Arg Gln Glu Lys Ala Leu Ser Ala Ile Ala Glu Leu Leu1 5 10 15Thr Ser Glu His Glu Arg 2018512PRTArtificial SequenceSynthetic Peptide 185Leu Ser Pro Glu Tyr Lys Gln Asn Glu Ile Asn Lys1 5 1018614PRTArtificial SequenceSynthetic Peptide 186Ser Pro Gly Thr Gly Ala Gly Leu Ala Glu Lys Ser Asp Arg1 5 1018715PRTArtificial SequenceSynthetic Peptide 187Arg Ser Pro Gly Thr Gly Ala Gly Leu Ala Glu Lys Ser Asp Arg1 5 10 1518810PRTArtificial SequenceSynthetic Peptide 188Tyr Glu Lys Glu Val His His Asp Ile Arg1 5 1018919PRTArtificial SequenceSynthetic Peptide 189Val Tyr Asp Leu Thr Lys Phe Leu Glu Glu His Pro Gly Gly Glu Glu1 5 10 15Val Leu Arg19025PRTArtificial SequenceSynthetic Peptide 190Leu Trp Tyr Thr Val Asp Lys Ala Pro Asp Ala Trp Asp Tyr Ser Gln1 5 10 15Gly Phe Val Asn Glu Glu Met Ile Arg 20 2519121PRTArtificial SequenceSynthetic Peptide 191Ile Gly Ser Thr Pro Val Val Val Leu Ser Gly Leu Asn Thr Ile Lys1 5 10 15Gln Ala Leu Val Arg 201929PRTArtificial SequenceSynthetic Peptide 192Tyr Leu Pro Asn Pro Ala Leu Lys Arg1 519323PRTArtificial SequenceSynthetic Peptide 193Thr Val Gln Glu His Tyr Gln Asp Phe Asn Lys Asn Ser Ile Gln Asp1 5 10 15Ile Thr Ser Ala Leu Phe Lys 2019416PRTArtificial SequenceSynthetic Peptide 194His Ser Glu Asn Tyr Lys Asp Asn Gly Gly Leu Ile Pro Glu Glu Lys1 5 10 1519513PRTArtificial SequenceSynthetic Peptide 195Asp Thr Ser Leu Asn Gly Phe His Ile Pro Lys Glu Arg1 5 1019614PRTArtificial SequenceSynthetic Peptide 196Asp Ser His Lys Leu Glu Asp Phe Met Ile Gln Lys Val Lys1 5 1019714PRTArtificial SequenceSynthetic Peptide 197Val Lys Gln Asn Gln Ser Thr Leu Asp Pro Asn Ser Pro Arg1 5 1019832PRTArtificial SequenceSynthetic Peptide 198Leu Ser Gly Lys Leu Pro Pro Gly Pro Thr Pro Leu Pro Phe Val Gly1 5 10 15Asn Phe Leu Gln Leu Asn Thr Glu Gln Met Tyr Asn Ser Leu Met Lys 20 25 3019932PRTArtificial SequenceSynthetic Peptide 199Leu Ser Gly Lys Leu Pro Pro Gly Pro Thr Pro Leu Pro Phe Val Gly1 5 10 15Asn Phe Leu Gln Leu Asn Thr Glu Gln Met Tyr Asn Ser Leu Met Lys 20 25 3020032PRTArtificial SequenceSynthetic Peptide 200Leu Ser Gly Lys Leu Pro Pro Gly Pro Thr Pro Leu Pro Phe Val Gly1 5 10 15Asn Phe Leu Gln Leu Asn Thr Glu Gln Met Tyr Asn Ser Leu Met Lys 20 25 3020122PRTArtificial SequenceSynthetic Peptide 201His Leu Pro Gly Pro Gln Gln Gln Ala Phe Lys Glu Leu Gln Gly Leu1 5 10 15Glu Asp Phe Ile Thr Lys 202029PRTArtificial SequenceSynthetic Peptide 202Asn Arg Gln Pro Lys Tyr Glu Asp Arg1 520315PRTArtificial SequenceSynthetic Peptide 203Met Lys Met Pro Tyr Thr Glu Ala Val Ile His Glu Ile Gln Arg1 5 10 1520411PRTArtificial SequenceSynthetic Peptide 204Phe Phe Ser Asn Pro Lys Asp Phe Asn Pro Lys1 5 1020512PRTArtificial SequenceSynthetic Peptide 205Glu Leu Gln Gly Leu Glu Asp Phe Ile Thr Lys Lys1 5 1020615PRTArtificial SequenceSynthetic Peptide 206Thr Lys Met Pro Tyr Thr Asp Ala Val Ile His Glu Ile Gln Arg1 5 10 1520713PRTArtificial SequenceSynthetic Peptide 207Lys Ser Asp Tyr Phe Met Pro Phe Ser Thr Gly Lys Arg1 5 1020812PRTArtificial SequenceSynthetic Peptide 208Ser Asp Tyr Phe Met Pro Phe Ser Thr Gly Lys Arg1 5 1020926PRTArtificial SequenceSynthetic Peptide 209Lys Pro Thr Val Val Leu His Gly Tyr Glu Ala Val Lys Glu Ala Leu1 5 10 15Val Asp His Gly Glu Glu Phe Ala Gly Arg 20 2521037PRTArtificial SequenceSynthetic Peptide 210Gly Thr Thr Val Ile Thr Ser Leu Ser Ser Val Leu His Asp Ser Lys1 5 10 15Glu Phe Pro Asn Pro Glu Met Phe Asp Pro Gly His Phe Leu Asn Gly 20 25 30Asn Gly Asn Phe Lys 3521137PRTArtificial SequenceSynthetic Peptide 211Gly Thr Thr Val Val Thr Ser Leu Thr Ser Val Leu His Asp Ser Lys1 5 10 15Glu Phe Pro Asn Pro Glu Leu Phe Asp Pro Gly His Phe Leu Asp Ala 20 25 30Asn Gly Asn Phe Lys 3521211PRTArtificial SequenceSynthetic Peptide 212Asp Phe Ile Asp Tyr Tyr Leu Ile Lys Gln Lys1 5 1021320PRTArtificial SequenceSynthetic Peptide 213Tyr Ile Asp Leu Gly Pro Asn Gly Val Val His Glu Val Thr Cys Asp1 5 10 15Thr Lys Phe Arg 2021413PRTArtificial SequenceSynthetic Peptide 214Gly Lys Gly Ile Gly Phe Ser His Gly Asn Val Trp Lys1 5 1021514PRTArtificial SequenceSynthetic Peptide 215Val Gln Glu Glu Ala Gln Trp Leu Met Lys Glu Leu Lys Lys1 5 1021614PRTArtificial SequenceSynthetic Peptide 216Val Gln Glu Glu Ala Gln Trp Leu Met Lys Glu Leu Lys Lys1 5 1021713PRTArtificial SequenceSynthetic Peptide 217Val Gln Glu Glu Ala Gln Trp Leu Met Lys Glu Leu Lys1 5 1021813PRTArtificial SequenceSynthetic Peptide 218Val Gln Glu Glu Ala Gln Trp Leu Met Lys Glu Leu Lys1 5 1021913PRTArtificial SequenceSynthetic Peptide 219Val Gln Glu Glu Ala Gln Trp Leu Met Lys Glu Leu Lys1 5 1022014PRTArtificial SequenceSynthetic Peptide 220Val Gln Glu Glu Ala Gln Trp Leu Met Lys Glu Leu Lys Lys1 5 1022114PRTArtificial SequenceSynthetic Peptide 221Ile Lys Glu His Glu Glu Ser Leu Asp Val Thr Asn Pro Arg1 5 1022226PRTArtificial SequenceSynthetic Peptide 222Lys Pro Thr Val Val Leu His Gly Tyr Glu Ala Val Lys Glu Ala Leu1 5 10 15Val Asp His Gly Asp Val Phe Ala Gly Arg 20 252239PRTArtificial SequenceSynthetic Peptide 223Phe Leu Lys Asp Val Thr Gln Gln Lys1 522410PRTArtificial SequenceSynthetic Peptide 224Phe Leu Lys Asp Val Thr Gln Gln Lys Lys1 5 1022520PRTArtificial SequenceSynthetic Peptide 225His Gln Lys Ser Leu Asp Leu Ser Asn Pro Gln Asp Phe Ile Asp Tyr1 5 10 15Phe Leu Ile Lys 2022623PRTArtificial SequenceSynthetic Peptide 226Gly Ser Ile Leu Ile Pro Asn Met Ser Ser Val Leu Lys Asp Glu Thr1 5 10 15Val Trp Glu Lys Pro Leu Arg 2022723PRTArtificial SequenceSynthetic Peptide 227Gly Thr Thr Leu Ile Pro Asn Leu Ser Ser Val Leu Lys Asp Glu Thr1 5 10 15Val Trp Glu Lys Pro Leu Arg 2022823PRTArtificial SequenceSynthetic Peptide 228Gly Thr Thr Leu Ile Cys Asn Leu Ser Ser Val Leu Lys Asp Glu Thr1 5 10 15Val Trp Glu Lys Pro Leu Arg 202297PRTArtificial SequenceSynthetic Peptide 229Ser Leu Thr Lys Leu Ala Lys1 523011PRTArtificial SequenceSynthetic Peptide 230Ile Val Val Leu His Gly Tyr Lys Ala Val Lys1 5 1023112PRTArtificial SequenceSynthetic Peptide 231Arg Ile Val Val Leu His Gly Tyr Lys Ala Val Lys1 5 1023216PRTArtificial SequenceSynthetic Peptide 232Ala Val Lys Glu Val Leu Leu Asn His Lys Asn Glu Phe Ser Gly Arg1 5 10 1523313PRTArtificial SequenceSynthetic Peptide 233Glu Val Leu Leu Asn His Lys Asn Glu Phe Ser Gly Arg1 5 1023412PRTArtificial SequenceSynthetic Peptide 234Gly Asp Ile Pro Val Phe Gln Glu Tyr Lys Asn Lys1 5 1023513PRTArtificial SequenceSynthetic Peptide 235Asn Lys Gly Ile Ile Phe Asn Asn Gly Pro Thr Trp Lys1 5 1023614PRTArtificial SequenceSynthetic Peptide 236Gly Ile Ile Phe Asn Asn Gly Pro Thr Trp Lys Asp Val Arg1 5 1023712PRTArtificial SequenceSynthetic Peptide 237Asp Trp Gly Met Gly Lys Gln Gly Asn Glu Ala Arg1 5 1023812PRTArtificial SequenceSynthetic Peptide 238Asp Trp Gly Met Gly Lys Gln Gly Asn Glu Ala Arg1 5 1023911PRTArtificial SequenceSynthetic Peptide 239Glu Ala His Phe Leu Val Glu Glu Leu Lys Lys1 5 1024027PRTArtificial SequenceSynthetic Peptide 240Thr Lys Gly Gln Pro Phe Asp Pro Thr Phe Leu Ile Gly Cys Ala Pro1 5 10 15Cys Asn Val Ile Ala Asp Ile Leu Phe Asn Lys 20 2524114PRTArtificial SequenceSynthetic Peptide 241Ala Lys Glu His Leu Lys Ser Leu Asp Ile Asn Cys Pro Arg1 5 1024212PRTArtificial SequenceSynthetic Peptide 242Glu His Leu Lys Ser Leu Asp Ile Asn Cys Pro Arg1 5 1024314PRTArtificial SequenceSynthetic Peptide 243Asp Val Thr Asp Cys Leu Leu Ile Glu Met Glu Lys Glu Lys1 5 1024412PRTArtificial SequenceSynthetic Peptide 244Tyr Ser Asp Tyr Phe Lys Ala Phe Ser Ala Gly Lys1 5 1024513PRTArtificial SequenceSynthetic Peptide 245Tyr Ser Asp Tyr Phe Lys Ala Phe Ser Ala Gly Lys Arg1 5 1024622PRTArtificial SequenceSynthetic Peptide 246Ser Leu Val Asp Pro Lys Asp Ile Asp Leu Ser Pro Val Thr Ile Gly1 5 10 15Phe Gly Ser Ile Pro Arg 2024721PRTArtificial SequenceSynthetic Peptide 247Lys Gln Gly Ile Pro Gly Pro Thr Pro Leu Pro Phe Leu Gly Thr Val1 5 10 15Leu Asn Tyr Tyr Lys 2024815PRTArtificial SequenceSynthetic Peptide 248Phe Cys Lys Lys Asp Val Glu Leu Asn Gly Val Tyr Ile Pro Lys1 5 10 1524919PRTArtificial SequenceSynthetic Peptide 249Glu Asn Lys Gly Ser Ile Asp Pro Tyr Leu Tyr Met Pro Phe Gly Ile1 5 10 15Gly Pro Arg25019PRTArtificial SequenceSynthetic Peptide 250Glu Asn Lys Gly Ser Ile Asp Pro Tyr Leu Tyr Met Pro Phe Gly Ile1 5 10 15Gly Pro Arg25122PRTArtificial SequenceSynthetic Peptide 251Phe Ser Lys Glu Asn Lys Gly Ser Ile Asp Pro Tyr Leu Tyr Met Pro1 5 10 15Phe Gly Ile Gly Pro Arg 2025221PRTArtificial SequenceSynthetic Peptide 252Val Met Gln Asn Phe Ser Phe Gln Pro Cys Gln Glu Thr Gln Ile Pro1 5 10 15Leu Lys Leu Ser Arg 202536PRTArtificial SequenceSynthetic Peptide 253Phe Ser Lys Glu Asn Lys1 525422PRTArtificial SequenceSynthetic Peptide 254Phe Ser Lys Glu Asn Lys Gly Ser Ile Asp Pro Tyr Val Tyr Leu Pro1 5 10 15Phe Gly Ile Gly Pro Arg 2025513PRTArtificial SequenceSynthetic Peptide 255Gln Gly Ile Leu Gln Pro Glu Lys Pro Ile Val Leu Lys1 5 1025617PRTArtificial SequenceSynthetic Peptide 256Gln Gly Ile Leu Gln Pro Glu Lys Pro Ile Val Leu Lys Val Val Pro1 5 10 15Arg25722PRTArtificial SequenceSynthetic Peptide 257Glu Phe Gly Pro Val Gly Ile Met Ser Lys Ala Ile Ser Ile Ser Lys1 5 10 15Asp Glu Glu Trp Lys Arg 2025815PRTArtificial SequenceSynthetic Peptide 258Asn Val Leu Val Lys Glu Cys Phe Ser Val Phe Thr Asn Arg Arg1 5 10 1525913PRTArtificial SequenceSynthetic Peptide 259Ala Leu Leu Ser Pro Thr Phe Thr Ser Gly Lys Leu Lys1 5 1026013PRTArtificial SequenceSynthetic Peptide 260Gln Gly Leu Leu Gln Pro Glu Lys Pro Ile Val Leu Lys1 5 1026121PRTArtificial SequenceSynthetic Peptide 261Lys Gln Gly Ile Pro Gly Pro Lys Pro Leu Pro Phe Leu Gly Thr Val1 5 10 15Leu Asn Tyr Tyr Lys 2026220PRTArtificial SequenceSynthetic Peptide 262Gln Gly Ile Pro Gly Pro Lys Pro Leu Pro Phe Leu Gly Thr Val Leu1 5 10 15Asn Tyr Tyr Lys 2026314PRTArtificial SequenceSynthetic Peptide 263Asn Val Leu Val Lys Glu Cys Phe Ser Val Phe Thr Asn Arg1 5 1026420PRTArtificial SequenceSynthetic Peptide 264Leu Lys Glu Met Phe Pro Val Ile Glu Gln Tyr Gly Asp Ile Leu Val1 5 10 15Lys Tyr Leu Arg 2026518PRTArtificial SequenceSynthetic Peptide 265Glu Met Phe Pro Val Ile Glu Gln Tyr Gly Asp Ile Leu Val Lys Tyr1 5 10 15Leu Arg26620PRTArtificial SequenceSynthetic Peptide 266Leu Lys Glu Met Phe Pro Val Ile Glu Gln Tyr Gly Asp Ile Leu Val1 5 10 15Lys Tyr Leu Arg 2026717PRTArtificial SequenceSynthetic Peptide 267Leu Lys Glu Met Phe Pro Val Ile Glu Gln Tyr Gly Asp Ile Leu Val1 5 10 15Lys26817PRTArtificial SequenceSynthetic Peptide 268Leu Lys Glu Met Phe Pro Val Ile Glu Gln Tyr Gly Asp Ile Leu Val1 5 10 15Lys2698PRTArtificial SequenceSynthetic Peptide 269Asp Ser Ile Glu Phe Phe Lys Lys1 527012PRTArtificial SequenceSynthetic Peptide 270Gly Leu Trp Lys Phe Asp Met Glu Cys Tyr Glu Lys1 5 1027118PRTArtificial SequenceSynthetic Peptide 271Leu Ala Lys Gln Ala Cys Gln Leu Ala His Asp His Thr Asp Gly Val1 5 10 15Ile Lys27221PRTArtificial SequenceSynthetic Peptide 272Tyr Leu Gln Asp Asn Pro Ala Ser Gly Glu Lys Phe Ala Tyr Val Pro1 5 10 15Phe Gly Ala Gly Arg 2027328PRTArtificial SequenceSynthetic Peptide 273Leu Asp Phe Asn Pro Asp Arg Tyr Leu Gln Asp Asn Pro Ala Ser Gly1 5 10 15Glu Lys Phe Ala Tyr Val Pro Phe Gly Ala Gly Arg 20 2527419PRTArtificial SequenceSynthetic Peptide 274Ser Ile Asp Pro Ser Asp Gly Asn Thr Thr Glu Asn Ile Asn Lys Thr1 5 10 15Phe Asn Lys27519PRTArtificial SequenceSynthetic Peptide 275Val Val Gln Glu Asp Tyr Val Leu Lys Met Ala Ser Gly Gln Glu Tyr1 5 10 15Gln Ile Arg27620PRTArtificial SequenceSynthetic Peptide 276Gln Ala Thr Val Gly Asp Val Asn Thr Asp Arg Pro Gly Leu Leu Asp1 5 10 15Leu Lys Gly Lys 2027712PRTArtificial SequenceSynthetic Peptide 277Gln Met Gly Gln Pro Cys Asp Ala Tyr Gln Lys Arg1 5 1027810PRTArtificial SequenceSynthetic Peptide 278Ala Leu Tyr Lys Ala Ile Ser Val Pro Arg1 5 102797PRTArtificial SequenceSynthetic Peptide 279Trp Gln Leu Asp Lys Ile Lys1 52807PRTArtificial SequenceSynthetic Peptide 280Val Lys His Phe Glu Ala Arg1 528111PRTArtificial SequenceSynthetic Peptide 281Ser Gly Ala Val Gln Lys Cys Ser Ala Cys Arg1 5 1028219PRTArtificial SequenceSynthetic Peptide 282Val Ala Glu Leu Met Gly Gln Lys Leu Pro Ser Phe Gly Pro Tyr Leu1 5 10 15Glu Gln Arg28315PRTArtificial SequenceSynthetic Peptide 283Leu Gly Asp Lys Val Gln Phe Cys His Thr Asp Asp Asn Gln Lys1 5 10 152846PRTArtificial SequenceSynthetic Peptide 284Ile Ser Ile Gly Lys Lys1 528511PRTArtificial SequenceSynthetic Peptide 285Tyr Phe Asp Pro Ala Asn Gly Lys Phe Ser Lys1 5 1028611PRTArtificial SequenceSynthetic Peptide 286Phe Ser Lys Ser Ala Asn Ser Pro Asp Gly Lys1 5 1028726PRTArtificial SequenceSynthetic Peptide 287Thr Glu Pro Thr Ala Gln Gln Asn Leu Ala Leu Gln Leu Ala Glu Lys1 5 10 15Leu Gly Ser Leu Val Glu Asn Asn Glu Arg 20 2528823PRTArtificial SequenceSynthetic Peptide 288Glu Gln Pro Glu Lys Glu Pro Glu Leu Gln Gln Tyr Val Pro Gln Leu1 5 10 15Gln Asn Asn Thr Ile Leu Arg 2028914PRTArtificial SequenceSynthetic Peptide 289Gln Ser Val Tyr Glu Glu Lys Leu Lys Gln Phe Glu Glu Arg1 5 1029027PRTArtificial SequenceSynthetic Peptide 290Glu Asn Met Tyr Ala Val Gln Thr Leu Lys Asp Phe Gln Tyr Val Asp1 5

10 15Arg Asp Gly Lys Asp Gln Gly Val Asn Val Arg 20 2529117PRTArtificial SequenceSynthetic Peptide 291Cys Phe Gly Gly Leu Gln Lys Val Phe Glu His Ser Ser Val Glu Leu1 5 10 15Lys29225PRTArtificial SequenceSynthetic Peptide 292Tyr Gln Leu Gln Ser Gln Glu Asn Phe Glu Pro Phe Met Lys Ala Ile1 5 10 15Gly Leu Pro Glu Asp Leu Ile Gln Lys 20 2529313PRTArtificial SequenceSynthetic Peptide 293Ala Ile Gly Leu Pro Glu Asp Leu Ile Gln Lys Gly Lys1 5 1029415PRTArtificial SequenceSynthetic Peptide 294Gly Lys Asp Ile Lys Gly Val Ser Glu Ile Val His Glu Gly Lys1 5 10 1529513PRTArtificial SequenceSynthetic Peptide 295Asp Ile Lys Gly Val Ser Glu Ile Val His Glu Gly Lys1 5 1029611PRTArtificial SequenceSynthetic Peptide 296Gly Val Ser Glu Ile Val His Glu Gly Lys Lys1 5 1029712PRTArtificial SequenceSynthetic Peptide 297Val Lys Ala Val Val Lys Leu Glu Gly Asp Asn Lys1 5 1029810PRTArtificial SequenceSynthetic Peptide 298Ala Val Val Lys Leu Glu Gly Asp Asn Lys1 5 102999PRTArtificial SequenceSynthetic Peptide 299Met Val Thr Thr Phe Lys Gly Ile Lys1 530010PRTArtificial SequenceSynthetic Peptide 300Trp Glu Glu Ile Gln Lys His Asn Leu Arg1 5 1030119PRTArtificial SequenceSynthetic Peptide 301Phe Leu Lys Pro Leu Leu Ile Gly Glu Leu Ala Pro Glu Glu Pro Ser1 5 10 15Leu Asp Arg30220PRTArtificial SequenceSynthetic Peptide 302Arg Pro Met Gly Gln Met Arg Pro Asp Asn Ser Lys Pro Pro Val Tyr1 5 10 15Gly Ala Cys Arg 2030317PRTArtificial SequenceSynthetic Peptide 303Ala Lys Ile Arg Gly Ser Tyr Leu Thr Val Thr Leu Gln Arg Pro Thr1 5 10 15Lys30417PRTArtificial SequenceSynthetic Peptide 304Gln Ile Leu Glu Glu Asn Tyr Gly Gln Lys Asp Pro Glu Lys Val Ala1 5 10 15Arg30517PRTArtificial SequenceSynthetic Peptide 305Gln Ile Leu Glu Glu Asn Tyr Gly Gln Lys Asp Pro Glu Lys Val Ala1 5 10 15Arg30629PRTArtificial SequenceSynthetic Peptide 306Val Leu Val Ile Gly Leu Gly Asn Ser Gly Cys Asp Ile Ala Ala Glu1 5 10 15Leu Ser His Val Ala Gln Lys Val Thr Ile Ser Ser Arg 20 2530711PRTArtificial SequenceSynthetic Peptide 307Asn Asn Tyr Met Glu Lys Gln Met Asn Gln Arg1 5 1030830PRTArtificial SequenceSynthetic Peptide 308Ser Lys Ala Glu Glu Val Val Ser Phe Val Lys Lys Asn Val Leu Val1 5 10 15Thr Gly Gly Phe Phe Gly Gly Phe Leu Leu Gly Met Ala Ser 20 25 3030930PRTArtificial SequenceSynthetic Peptide 309Thr Phe Thr Thr Gln Glu Thr Ile Thr Asn Ala Glu Thr Ala Lys Glu1 5 10 15Trp Phe Leu Glu Ala Ala Lys Asp Pro Ser Ala Val Ala Lys 20 25 3031019PRTArtificial SequenceSynthetic Peptide 310Gly Ala Ala Gln Asn Ile Ile Pro Ala Ser Thr Gly Ala Ala Lys Ala1 5 10 15Val Gly Lys31112PRTArtificial SequenceSynthetic Peptide 311Leu Glu Lys Pro Ala Lys Tyr Asp Asp Ile Lys Lys1 5 1031233PRTArtificial SequenceSynthetic Peptide 312Thr Leu Lys Asn Gly Thr Thr Thr Ala Cys Tyr Phe Gly Thr Ile His1 5 10 15Thr Asp Ser Ser Leu Ile Leu Ala Glu Ile Thr Asp Lys Phe Gly Gln 20 25 30Arg31317PRTArtificial SequenceSynthetic Peptide 313Val Ser Lys Ala Ser Ala Asp Leu Met Ser Tyr Cys Glu Glu His Ala1 5 10 15Arg31413PRTArtificial SequenceSynthetic Peptide 314Lys Asp Pro Asn Lys Leu Val Leu Cys Glu Val Phe Lys1 5 103156PRTArtificial SequenceSynthetic Peptide 315Tyr Ala Leu Lys Glu Arg1 531614PRTArtificial SequenceSynthetic Peptide 316Val Leu Lys Ser His Gly Gln Asp Tyr Leu Val Gly Asn Arg1 5 1031724PRTArtificial SequenceSynthetic Peptide 317Ser Asp Gly Ser Leu Met Phe Gln Gln Val Pro Met Val Glu Ile Asp1 5 10 15Gly Met Lys Leu Val Gln Thr Lys 2031824PRTArtificial SequenceSynthetic Peptide 318Ser Asp Gly Ser Leu Met Phe Gln Gln Val Pro Met Val Glu Ile Asp1 5 10 15Gly Met Lys Leu Val Gln Thr Lys 2031924PRTArtificial SequenceSynthetic Peptide 319Ser Asp Gly Ser Leu Met Phe Gln Gln Val Pro Met Val Glu Ile Asp1 5 10 15Gly Met Lys Leu Val Gln Thr Lys 203209PRTArtificial SequenceSynthetic Peptide 320Ile Ser Ala Tyr Met Lys Ser Ser Arg1 532119PRTArtificial SequenceSynthetic Peptide 321Phe Lys Leu Gly Leu Asp Phe Pro Asn Leu Pro Tyr Leu Ile Asp Gly1 5 10 15Ser His Lys32226PRTArtificial SequenceSynthetic Peptide 322Leu Gly Leu Asp Phe Pro Asn Leu Pro Tyr Leu Ile Asp Gly Ser His1 5 10 15Lys Ile Thr Gln Ser Asn Ala Ile Leu Arg 20 2532320PRTArtificial SequenceSynthetic Peptide 323Ala Leu Pro Gly His Leu Lys Pro Phe Glu Thr Leu Leu Ser Gln Asn1 5 10 15Gln Gly Gly Lys 2032413PRTArtificial SequenceSynthetic Peptide 324Ala Lys Asp Met Pro Pro Leu Met Asp Pro Ala Leu Lys1 5 1032518PRTArtificial SequenceSynthetic Peptide 325Ala Met Leu Glu Ala His Pro Lys Val Val Ala His Tyr Pro Val Glu1 5 10 15Val Arg32618PRTArtificial SequenceSynthetic Peptide 326Ala Met Leu Glu Ala His Pro Lys Val Val Ala His Tyr Pro Val Glu1 5 10 15Val Arg32714PRTArtificial SequenceSynthetic Peptide 327Ser His Tyr Lys Val Gly Glu Asn Ala Asp Ser Gln Ile Lys1 5 1032828PRTArtificial SequenceSynthetic Peptide 328Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile Gln Ala Val Leu1 5 10 15Leu Pro Lys Lys Thr Glu Ser His His Lys Pro Lys 20 2532915PRTArtificial SequenceSynthetic Peptide 329Lys Ser Ser Ala Thr Val Gly Pro Lys Ala Pro Ala Val Gly Lys1 5 10 1533011PRTArtificial SequenceSynthetic Peptide 330Pro Glu Pro Ala Lys Ser Ala Pro Ala Pro Lys1 5 103319PRTArtificial SequenceSynthetic Peptide 331Ser Asn Ile Lys Ala Ala Trp Gly Lys1 533220PRTArtificial SequenceSynthetic Peptide 332Ala Ala Trp Gly Lys Ile Gly Gly His Gly Ala Glu Tyr Gly Ala Glu1 5 10 15Ala Leu Glu Arg 2033325PRTArtificial SequenceSynthetic Peptide 333Met Phe Ala Ser Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe Asp1 5 10 15Val Ser His Gly Ser Ala Gln Val Lys 20 2533425PRTArtificial SequenceSynthetic Peptide 334Met Phe Ala Ser Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe Asp1 5 10 15Val Ser His Gly Ser Ala Gln Val Lys 20 2533520PRTArtificial SequenceSynthetic Peptide 335Thr Tyr Phe Pro His Phe Asp Val Ser His Gly Ser Ala Gln Val Lys1 5 10 15Gly His Gly Lys 2033631PRTArtificial SequenceSynthetic Peptide 336Val Ala Asp Ala Leu Ala Ser Ala Ala Gly His Leu Asp Asp Leu Pro1 5 10 15Gly Ala Leu Ser Ala Leu Ser Asp Leu His Ala His Lys Leu Arg 20 25 3033732PRTArtificial SequenceSynthetic Peptide 337Lys Val Ala Asp Ala Leu Ala Ser Ala Ala Gly His Leu Asp Asp Leu1 5 10 15Pro Gly Ala Leu Ser Ala Leu Ser Asp Leu His Ala His Lys Leu Arg 20 25 3033822PRTArtificial SequenceSynthetic Peptide 338Ser Ala Val Ser Cys Leu Trp Ala Lys Val Asn Pro Asp Glu Val Gly1 5 10 15Gly Glu Ala Leu Gly Arg 2033921PRTArtificial SequenceSynthetic Peptide 339Tyr Phe Asp Ser Phe Gly Asp Leu Ser Ser Ala Ser Ala Ile Met Gly1 5 10 15Asn Pro Lys Val Lys 2034028PRTArtificial SequenceSynthetic Peptide 340Asn Leu Asp Asn Leu Lys Gly Thr Phe Ala Ser Leu Ser Glu Leu His1 5 10 15Cys Asp Lys Leu His Val Asp Pro Glu Asn Phe Arg 20 2534122PRTArtificial SequenceSynthetic Peptide 341Ala Ala Val Ser Cys Leu Trp Gly Lys Val Asn Ser Asp Glu Val Gly1 5 10 15Gly Glu Ala Leu Gly Arg 2034221PRTArtificial SequenceSynthetic Peptide 342Tyr Phe Asp Ser Phe Gly Asp Leu Ser Ser Ala Ser Ala Ile Met Gly1 5 10 15Asn Ala Lys Val Lys 2034338PRTArtificial SequenceSynthetic Peptide 343Val Ile Thr Ala Phe Asn Asp Gly Leu Asn His Leu Asp Ser Leu Lys1 5 10 15Gly Thr Phe Ala Ser Leu Ser Glu Leu His Cys Asp Lys Leu His Val 20 25 30Asp Pro Glu Asn Phe Arg 3534433PRTArtificial SequenceSynthetic Peptide 344Ile Leu Pro Ser Val Ser His Lys Pro Phe Glu Ser Ile Asp Gln Gly1 5 10 15His Val Thr His Asn Trp Asp Glu Val Gly Pro Asp Pro Asn Gln Leu 20 25 30Arg3459PRTArtificial SequenceSynthetic Peptide 345Phe Gln Gly Lys Leu Phe Ala Cys Lys1 534613PRTArtificial SequenceSynthetic Peptide 346Met Phe Val Gly Gly Leu Ser Trp Asp Thr Ser Lys Lys1 5 1034713PRTArtificial SequenceSynthetic Peptide 347Met Phe Val Gly Gly Leu Ser Trp Asp Thr Ser Lys Lys1 5 1034818PRTArtificial SequenceSynthetic Peptide 348Val Ala Gln Pro Lys Glu Val Tyr Gln Gln Gln Gln Tyr Gly Ser Gly1 5 10 15Gly Arg34911PRTArtificial SequenceSynthetic Peptide 349Glu Val Val Ser His Val Ile Lys Gln Gly Lys1 5 1035014PRTArtificial SequenceSynthetic Peptide 350Ile Val Arg Glu Pro Trp Val Glu Gln Asp Lys Phe Gly Lys1 5 1035116PRTArtificial SequenceSynthetic Peptide 351Ile Val Arg Glu Pro Trp Val Glu Gln Asp Lys Phe Gly Lys Val Lys1 5 10 1535219PRTArtificial SequenceSynthetic Peptide 352Val Lys Phe Ala Val Leu Gln Thr Tyr Gly Asp Thr Thr His Thr Leu1 5 10 15Val Glu Lys35316PRTArtificial SequenceSynthetic Peptide 353Ile Val Arg Glu Pro Trp Val Glu Gln Asp Lys Phe Gly Lys Val Lys1 5 10 1535422PRTArtificial SequenceSynthetic Peptide 354Ser Ile Val Val Thr Asn Tyr Glu Glu Ser Ile Lys Met Pro Ile Asn1 5 10 15Glu Pro Ala Pro Gly Arg 2035523PRTArtificial SequenceSynthetic Peptide 355Lys Lys Ser Gln Ile Gln Glu Tyr Val Asp Tyr Asn Gly Gly Ala Gly1 5 10 15Val Gln His Ile Ala Leu Lys 2035623PRTArtificial SequenceSynthetic Peptide 356Lys Lys Ser Gln Ile Gln Glu Tyr Val Asp Tyr Asn Gly Gly Ala Gly1 5 10 15Val Gln His Ile Ala Leu Lys 2035730PRTArtificial SequenceSynthetic Peptide 357Ser Gln Ile Gln Glu Tyr Val Asp Tyr Asn Gly Gly Ala Gly Val Gln1 5 10 15His Ile Ala Leu Lys Thr Glu Asp Ile Ile Thr Ala Ile Arg 20 25 3035818PRTArtificial SequenceSynthetic Peptide 358Glu Arg Gly Thr Glu Phe Leu Ala Ala Pro Ser Ser Tyr Tyr Lys Leu1 5 10 15Leu Arg35925PRTArtificial SequenceSynthetic Peptide 359His Asn His Gln Gly Phe Gly Ala Gly Asn Phe Asn Ser Leu Phe Lys1 5 10 15Ala Phe Glu Glu Glu Gln Ala Leu Arg 20 2536018PRTArtificial SequenceSynthetic Peptide 360Asn Leu Gly Glu Ile Leu Lys Ala Ala Gly Cys Asp Phe Asn Asn Val1 5 10 15Val Lys36110PRTArtificial SequenceSynthetic Peptide 361Val Gln Val Glu Tyr Lys Gly Glu Thr Lys1 5 103628PRTArtificial SequenceSynthetic Peptide 362Leu Ser Lys Glu Asp Ile Glu Arg1 536314PRTArtificial SequenceSynthetic Peptide 363Met Val Gln Glu Ala Glu Lys Tyr Lys Ala Glu Asp Glu Lys1 5 1036416PRTArtificial SequenceSynthetic Peptide 364Met Val Gln Glu Ala Glu Lys Tyr Lys Ala Glu Asp Glu Lys Gln Arg1 5 10 1536516PRTArtificial SequenceSynthetic Peptide 365Met Val Gln Glu Ala Glu Lys Tyr Lys Ala Glu Asp Glu Lys Gln Arg1 5 10 1536628PRTArtificial SequenceSynthetic Peptide 366Ile Leu Asp Lys Cys Asn Glu Ile Ile Ser Trp Leu Asp Lys Asn Gln1 5 10 15Thr Ala Glu Lys Glu Glu Phe Glu His Gln Gln Lys 20 2536732PRTArtificial SequenceSynthetic Peptide 367Ile Leu Asp Lys Cys Asn Glu Ile Ile Ser Trp Leu Asp Lys Asn Gln1 5 10 15Thr Ala Glu Lys Glu Glu Phe Glu His Gln Gln Lys Glu Leu Glu Lys 20 25 303686PRTArtificial SequenceSynthetic Peptide 368Ile Pro Lys Ile Gln Lys1 536911PRTArtificial SequenceSynthetic Peptide 369Ser Glu Glu Gly Leu Gln Lys Val Val Ser Arg1 5 1037020PRTArtificial SequenceSynthetic Peptide 370Ile Lys Glu Glu Tyr Glu Val Ala Glu Met Gly Ala Pro His Gly Ser1 5 10 15Ala Ser Val Arg 2037120PRTArtificial SequenceSynthetic Peptide 371Ile Lys Glu Glu Tyr Glu Val Ala Glu Met Gly Ala Pro His Gly Ser1 5 10 15Ala Ser Val Arg 2037223PRTArtificial SequenceSynthetic Peptide 372Lys Lys Thr Ser Glu Glu Ile Leu Gln His Leu Gln Asn Ile Val Asp1 5 10 15Phe Gly Lys Asn Val Met Lys 2037332PRTArtificial SequenceSynthetic Peptide 373Ala Trp Val Asn Gln Leu Glu Thr Gln Thr Gly Glu Ala Ser Lys Leu1 5 10 15Pro Tyr Asp Val Thr Thr Glu Gln Ala Leu Thr Tyr Pro Glu Val Lys 20 25 3037416PRTArtificial SequenceSynthetic Peptide 374Thr Pro Glu Glu Gly Lys Gln Ser Gln Ala Val Ile Glu Asp Ala Arg1 5 10 1537510PRTArtificial SequenceSynthetic Peptide 375Asn Ile Glu Val Pro Phe Lys Pro Ala Arg1 5 1037619PRTArtificial SequenceSynthetic Peptide 376Val Ser Thr Asn Gly Ser Glu Asp Pro Glu Val Ala Ala Ser Gly Glu1 5 10 15Asn Lys Arg37730PRTArtificial SequenceSynthetic Peptide 377Phe Ile Tyr Gly Asn Gln Asp Leu Phe Ala Thr Ser Gln Asn Lys Glu1 5 10 15Phe Asp Pro Leu Gly Pro Leu Pro Pro Gly Trp Glu Lys Arg 20 25 3037813PRTArtificial SequenceSynthetic Peptide 378Val Thr Phe Asn Ser Ala Leu Ala Gln Lys Glu Ala Lys1 5 1037921PRTArtificial SequenceSynthetic Peptide 379Gly Lys Asn Cys Glu Leu Leu Leu Val Val Pro Glu Glu Val Glu Ala1 5 10 15His Gln Ser Trp Arg 2038013PRTArtificial SequenceSynthetic Peptide 380Ile Glu Glu His Asn Ala Lys Gln Pro Leu Pro Gln Lys1 5 1038116PRTArtificial SequenceSynthetic Peptide 381Ala Val Ala Lys Gln Gln Asn Val Gln Ser Thr Ser Gln Asp Glu Lys1 5 10 1538210PRTArtificial SequenceSynthetic Peptide 382Thr Tyr Ser Ala Lys Leu Asp Asn Ala Arg1 5 1038313PRTArtificial SequenceSynthetic Peptide 383Lys Glu Ser Asp Leu Ser Gly Ala Gln Ile Lys Leu Arg1 5 1038413PRTArtificial SequenceSynthetic Peptide 384Ile Pro Glu Lys Glu Pro Pro Pro Pro Tyr Leu Pro Ala1 5 1038529PRTArtificial SequenceSynthetic Peptide 385Val Ile Asp Leu Val Asn Lys Glu Asp Val Gln Ile Ser Thr Thr Gln1 5 10 15Val Ala Glu Ile Val Ala Thr Leu Glu Lys Glu Glu Lys 20 253868PRTArtificial SequenceSynthetic Peptide 386Leu Leu Glu Glu Ala Leu Lys Lys1 538721PRTArtificial SequenceSynthetic Peptide 387Met Gln Ala Asn Asn Ala Lys Ala Val Ser Ala Arg Thr Glu Ala Ile1 5 10 15Lys Ala Leu Val Lys

2038820PRTArtificial SequenceSynthetic Peptide 388Ser Tyr Leu Leu Glu Lys Ile Lys Glu His Glu Glu Ser Leu Asp Val1 5 10 15Thr Asn Pro Arg 2038915PRTArtificial SequenceSynthetic Peptide 389Lys His Met Pro Tyr Thr Asn Ala Met Val His Glu Val Gln Arg1 5 10 1539020PRTArtificial SequenceSynthetic Peptide 390Tyr Val Asp Leu Gly Pro Thr Ser Leu Val His Glu Val Thr Cys Asp1 5 10 15Thr Lys Phe Arg 2039114PRTArtificial SequenceSynthetic Peptide 391Asp Gln Pro Gln His Leu Glu Lys Ile Thr Cys Gln Gln Arg1 5 1039210PRTArtificial SequenceSynthetic Peptide 392Thr Leu Leu Lys Glu Ile Cys Leu Arg Asn1 5 1039323PRTArtificial SequenceSynthetic Peptide 393Thr Ala Ala Lys Gly Glu Ala Thr Ala Glu Arg Pro Gly Glu Ala Ala1 5 10 15Val Ala Ser Ser Pro Ser Lys 2039414PRTArtificial SequenceSynthetic Peptide 394Gln Asp Pro Asn Ala Lys Val Ala Cys Glu Thr Val Cys Lys1 5 1039513PRTArtificial SequenceSynthetic Peptide 395Asp Thr Ile Lys His Ile Gly Tyr Asp Asp Ser Ala Lys1 5 1039614PRTArtificial SequenceSynthetic Peptide 396His Ile Gly Tyr Asp Asp Ser Ala Lys Gly Phe Asp Phe Lys1 5 1039719PRTArtificial SequenceSynthetic Peptide 397Glu Leu Leu Glu Val Val Asn Lys Asn Phe Asp Leu Arg Pro Gly Val1 5 10 15Ile Val Arg39811PRTArtificial SequenceSynthetic Peptide 398Asp Leu Asp Leu Lys Lys Pro Ile Tyr Gln Lys1 5 1039915PRTArtificial SequenceSynthetic Peptide 399Lys Pro Ile Tyr Gln Lys Thr Ala Cys Tyr Gly His Phe Gly Arg1 5 10 1540020PRTArtificial SequenceSynthetic Peptide 400Asp Leu Asp Leu Lys Lys Pro Ile Tyr Gln Lys Thr Ala Cys Tyr Gly1 5 10 15His Phe Gly Arg 2040116PRTArtificial SequenceSynthetic Peptide 401Ser Asp Val Tyr Tyr Phe Ser Pro Ser Gly Lys Lys Phe Arg Ser Lys1 5 10 1540220PRTArtificial SequenceSynthetic Peptide 402Glu Gly Lys Glu Asp Glu Ala Ser Thr Asp Val Asp Glu Lys Pro Lys1 5 10 15Glu Thr Met Lys 2040320PRTArtificial SequenceSynthetic Peptide 403Glu Gly Lys Glu Asp Glu Ala Ser Thr Asp Val Asp Glu Lys Pro Lys1 5 10 15Glu Thr Met Lys 2040414PRTArtificial SequenceSynthetic Peptide 404Trp Leu Pro Val Gly Pro His Ile Met Gly Lys Ala Val Lys1 5 1040514PRTArtificial SequenceSynthetic Peptide 405Trp Leu Pro Val Gly Pro His Ile Met Gly Lys Ala Val Lys1 5 1040619PRTArtificial SequenceSynthetic Peptide 406Val Phe Ala Asn Pro Glu Asp Cys Ala Gly Phe Gly Lys Gly Glu Asn1 5 10 15Ala Lys Lys40719PRTArtificial SequenceSynthetic Peptide 407Val Phe Ala Asn Pro Glu Asp Cys Ala Gly Phe Gly Lys Gly Glu Asn1 5 10 15Ala Lys Lys40814PRTArtificial SequenceSynthetic Peptide 408Glu Leu Gln Lys Ala Asn Gln Glu Gln Tyr Ala Glu Gly Lys1 5 1040929PRTArtificial SequenceSynthetic Peptide 409Leu Cys Asp Pro Ser Glu Gln Ala Leu Tyr Gly Lys Leu Pro Ile Phe1 5 10 15Gly Gln Tyr Phe Ala Leu Glu Asn Pro Gly Thr Ile Arg 20 2541038PRTArtificial SequenceSynthetic Peptide 410Asp Tyr His Phe Val Ser Arg Gln Ala Phe Glu Ala Asp Ile Ala Ala1 5 10 15Gly Lys Phe Ile Glu His Gly Glu Phe Glu Lys Asn Leu Tyr Gly Thr 20 25 30Ser Ile Asp Ser Val Arg 3541122PRTArtificial SequenceSynthetic Peptide 411Met Asp Pro Asn Cys Ser Cys Ala Ser Asp Gly Ser Cys Ser Cys Ala1 5 10 15Gly Ala Cys Lys Cys Lys 2041219PRTArtificial SequenceSynthetic Peptide 412Ile His Lys Thr Ser Asn Pro Trp Gln Ser Pro Ser Gly Thr Leu Pro1 5 10 15Ala Leu Arg41312PRTArtificial SequenceSynthetic Peptide 413Gln Met Asn Asp Glu Lys Thr Ala Ala Asp Tyr Lys1 5 1041426PRTArtificial SequenceSynthetic Peptide 414Lys Val Gln Phe Ser Gln His Pro Ala Ala Ala Lys Leu Pro Leu Glu1 5 10 15Gln Ser Glu Asp Pro Ala Gly Leu Ile Arg 20 2541525PRTArtificial SequenceSynthetic Peptide 415Val Gln Phe Ser Gln His Pro Ala Ala Ala Lys Leu Pro Leu Glu Gln1 5 10 15Ser Glu Asp Pro Ala Gly Leu Ile Arg 20 2541617PRTArtificial SequenceSynthetic Peptide 416Gln Gly Gly Leu Gly Pro Met Asn Ile Pro Leu Ile Ser Asp Pro Lys1 5 10 15Arg41717PRTArtificial SequenceSynthetic Peptide 417Gln His Tyr Ile Asp Leu Lys Asp Arg Pro Phe Phe Pro Gly Leu Val1 5 10 15Lys41827PRTArtificial SequenceSynthetic Peptide 418Ala Glu Ser Lys Ala Pro Ala Gly Gly Gln Asn Asn Pro Ser Ser Ser1 5 10 15Pro Ser Gly Ser Thr Val Ser Gln Ala Ser Lys 20 254198PRTArtificial SequenceSynthetic Peptide 419Ser Phe Asn Gly Ser Leu Lys Lys1 542012PRTArtificial SequenceSynthetic Peptide 420Gly Ala Lys Glu His Gly Ala Val Ala Val Glu Arg1 5 1042141PRTArtificial SequenceSynthetic Peptide 421Ser Thr Gln Thr Ala Pro Ser Ser Ala Pro Ser Thr Gly Gln Lys Ser1 5 10 15Pro Arg Val Asn Pro Pro Val Pro Lys Ser Gly Ser Ser Gln Ala Lys 20 25 30Ala Leu Gln Pro Pro Val Thr Glu Lys 35 4042216PRTArtificial SequenceSynthetic Peptide 422Glu Pro Lys Glu Leu Lys Leu Ser Val Pro Met Pro Tyr Met Leu Lys1 5 10 154239PRTArtificial SequenceSynthetic Peptide 423Lys Phe Glu Gln Met Lys Gln Asp Arg1 542411PRTArtificial SequenceSynthetic Peptide 424Glu Glu Val Gly Ala Leu Ala Lys Val Leu Arg1 5 1042513PRTArtificial SequenceSynthetic Peptide 425Ser Lys Pro Val Leu Gly Ser Ile Ile Lys Ser Leu Arg1 5 1042632PRTArtificial SequenceSynthetic Peptide 426Thr Ile Gln Glu Leu Asp Arg Phe Ala Asn Gln Ile Leu Ser Tyr Gly1 5 10 15Ala Glu Leu Asp Ala Asp His Pro Gly Phe Lys Asp Pro Val Tyr Arg 20 25 3042725PRTArtificial SequenceSynthetic Peptide 427Ala Gly Glu Ile Lys Gly Phe Thr Gly Ile Asp Ser Asp Tyr Glu Lys1 5 10 15Pro Glu Thr Pro Glu Cys Val Leu Lys 20 2542814PRTArtificial SequenceSynthetic Peptide 428Trp Met Ser Glu Glu Asp Phe Glu Lys Ala Phe Asn Ala Arg1 5 1042914PRTArtificial SequenceSynthetic Peptide 429Trp Met Ser Glu Glu Asp Phe Glu Lys Ala Phe Asn Ala Arg1 5 1043012PRTArtificial SequenceSynthetic Peptide 430Ser Glu Ala Thr Ala Ala Ala Glu His Lys Gly Lys1 5 1043110PRTArtificial SequenceSynthetic Peptide 431Gln Gln Asn Gln His Pro Glu Lys Pro Arg1 5 1043224PRTArtificial SequenceSynthetic Peptide 432Glu Ala Ala Leu Asn Asp Lys Lys Pro Gly Pro Gly Met Asn Gly Ala1 5 10 15Val Glu Pro Cys Ala Gln Pro Arg 2043313PRTArtificial SequenceSynthetic Peptide 433Ala Glu Thr Ala Ala Lys His Gly Glu Ala Gln Val Lys1 5 1043412PRTArtificial SequenceSynthetic Peptide 434Glu Lys Gln Ala Ala Leu Glu Glu Glu Gln Ala Arg1 5 1043520PRTArtificial SequenceSynthetic Peptide 435Gly Ala Ile Gln Leu Gly Ile Gly Tyr Thr Val Gly Asn Leu Ser Ser1 5 10 15Lys Pro Glu Arg 2043610PRTArtificial SequenceSynthetic Peptide 436Arg Ser Met Ser Ser Trp Lys Leu Ser Lys1 5 1043714PRTArtificial SequenceSynthetic Peptide 437Ala Ala Ala Gln Tyr Ser Ser Gln Lys Ser Val Glu Glu Arg1 5 1043813PRTArtificial SequenceSynthetic Peptide 438Met Thr Ile Met Glu Gln Lys Tyr Glu Gly Glu Tyr Arg1 5 1043921PRTArtificial SequenceSynthetic Peptide 439Glu Gly Gly Trp Asp Ser Val Gln Asp Trp Met Asp Val Leu Ser Gly1 5 10 15Gly Glu Lys Gln Arg 2044018PRTArtificial SequenceSynthetic Peptide 440Leu Gln Pro Ile Ala Leu Ser Cys Val Leu Asn Ile Gly Ala Cys Lys1 5 10 15Leu Lys44119PRTArtificial SequenceSynthetic Peptide 441Ser Ser Phe Phe Val Asn Gly Leu Thr Leu Gly Gly Gln Lys Cys Ser1 5 10 15Val Ile Arg44224PRTArtificial SequenceSynthetic Peptide 442Ser Val His Lys Val Glu Pro Ile Thr Lys His Ile Gly Leu Val Tyr1 5 10 15Ser Gly Met Gly Pro Asp Tyr Arg 2044311PRTArtificial SequenceSynthetic Peptide 443Ala Arg Tyr Glu Ala Ala Asn Trp Lys Tyr Lys1 5 1044414PRTArtificial SequenceSynthetic Peptide 444Ala Thr Ala Gly Ala Tyr Ile Ala Ser Gln Thr Val Lys Lys1 5 1044510PRTArtificial SequenceSynthetic Peptide 445Tyr Ile Ile Asn Val Lys Gln Phe Ala Lys1 5 1044620PRTArtificial SequenceSynthetic Peptide 446Ile Ile Asn Ala Asp Ser Glu Asp Pro Lys Tyr Ile Ile Asn Val Lys1 5 10 15Gln Phe Ala Lys 204479PRTArtificial SequenceSynthetic Peptide 447Asp Ala Phe Ala Leu Ala Lys Glu Lys1 544825PRTArtificial SequenceSynthetic Peptide 448Asp Ala Phe Ala Leu Ala Lys Glu Lys Ala Pro Ser Ile Ile Phe Ile1 5 10 15Asp Glu Leu Asp Ala Ile Gly Thr Lys 20 2544920PRTArtificial SequenceSynthetic Peptide 449Ser Glu Asn Asp Leu Lys Ala Leu Gln Ser Val Gly Gln Ile Val Gly1 5 10 15Glu Val Leu Lys 2045021PRTArtificial SequenceSynthetic Peptide 450Ala Val Ala Ser Gln Leu Asp Cys Asn Phe Leu Lys Val Val Ser Ser1 5 10 15Ser Ile Val Asp Lys 2045128PRTArtificial SequenceSynthetic Peptide 451Ala Val Ala Ser Gln Leu Asp Cys Asn Phe Leu Lys Val Val Ser Ser1 5 10 15Ser Ile Val Asp Lys Tyr Ile Gly Glu Ser Ala Arg 20 2545217PRTArtificial SequenceSynthetic Peptide 452Ser Ser Asp Glu Ala Val Ile Leu Cys Lys Thr Ala Ile Gly Ala Leu1 5 10 15Lys45318PRTArtificial SequenceSynthetic Peptide 453Ile Ile Ala Phe Val Gly Ser Pro Val Glu Asp Asn Glu Lys Asp Leu1 5 10 15Val Lys45412PRTArtificial SequenceSynthetic Peptide 454Asn Phe Lys Val Met Ile Tyr Gln Asp Glu Val Lys1 5 1045511PRTArtificial SequenceSynthetic Peptide 455Tyr Glu Tyr Gly Ile Phe Asn Gln Lys Ile Arg1 5 1045615PRTArtificial SequenceSynthetic Peptide 456Ala Trp Asn Thr Met Val Leu Lys Asn Ile Ala Ala Ser Gly Lys1 5 10 1545715PRTArtificial SequenceSynthetic Peptide 457Ala Trp Asn Thr Met Val Leu Lys Asn Ile Ala Ala Ser Gly Lys1 5 10 1545819PRTArtificial SequenceSynthetic Peptide 458Thr Ala Ser Asn Val Glu Glu Ala Phe Ile Asn Thr Ala Lys Glu Ile1 5 10 15Tyr Glu Lys45910PRTArtificial SequenceSynthetic Peptide 459Lys Glu Asn Pro Leu Gln Phe Lys Phe Arg1 5 1046019PRTArtificial SequenceSynthetic Peptide 460Ile Ala Gln Asp Leu Glu Met Tyr Gly Val Asn Tyr Phe Glu Ile Lys1 5 10 15Asn Lys Lys46118PRTArtificial SequenceSynthetic Peptide 461Lys Lys Pro Ile Gln Glu Pro Glu Val Pro Gln Met Asp Ala Pro Ser1 5 10 15Val Lys4628PRTArtificial SequenceSynthetic Peptide 462Leu Asp Pro Glu Thr Gly Lys Arg1 546313PRTArtificial SequenceSynthetic Peptide 463Leu Gly Pro Gly Gln Leu Thr Trp Lys Asn Ser Glu Arg1 5 1046412PRTArtificial SequenceSynthetic Peptide 464Met Lys Gln Glu Pro Val Lys Pro Glu Glu Gly Arg1 5 1046517PRTArtificial SequenceSynthetic Peptide 465Glu Lys Thr Pro Pro Arg Pro Gln Gly Gln Arg Pro Glu Pro Glu Asn1 5 10 15Arg46615PRTArtificial SequenceSynthetic Peptide 466Asp Ser Val Lys Asp Lys Glu Lys Gly Lys His Asp Asp Gly Arg1 5 10 1546717PRTArtificial SequenceSynthetic Peptide 467Leu Lys Thr Pro Pro Arg Pro Gln Gly Gln Arg Pro Ala Pro Glu Ser1 5 10 15Arg46817PRTArtificial SequenceSynthetic Peptide 468Glu Lys Thr Cys Pro Leu Cys Arg Thr Val Ile Ser Glu Cys Ile Asn1 5 10 15Lys46910PRTArtificial SequenceSynthetic Peptide 469Ile Thr Val Lys Leu Thr Ile Gln Asn Arg1 5 1047020PRTArtificial SequenceSynthetic Peptide 470Lys Ser Ala Glu Phe Leu Leu His Met Leu Lys Asn Ala Glu Ser Asn1 5 10 15Ala Glu Leu Lys 2047116PRTArtificial SequenceSynthetic Peptide 471Glu Asn Lys Thr Ala Val Val Val Gly Thr Val Thr Asp Asp Val Arg1 5 10 1547216PRTArtificial SequenceSynthetic Peptide 472Lys Glu Glu Ile Ile Lys Thr Leu Ser Lys Glu Glu Glu Thr Lys Lys1 5 10 1547310PRTArtificial SequenceSynthetic Peptide 473Thr Leu Ser Lys Glu Glu Glu Thr Lys Lys1 5 1047410PRTArtificial SequenceSynthetic Peptide 474Thr Leu Ser Lys Glu Glu Glu Thr Lys Lys1 5 1047515PRTArtificial SequenceSynthetic Peptide 475Ala Pro Ala Lys Ala Gln Ala Ser Ala Pro Ala Gln Ala Pro Lys1 5 10 1547617PRTArtificial SequenceSynthetic Peptide 476Ala Gln Ala Ser Ala Pro Ala Gln Ala Pro Lys Gly Ala Gln Ala Pro1 5 10 15Lys47711PRTArtificial SequenceSynthetic Peptide 477Ile Gly Gln Gly Tyr Leu Ile Lys Asp Gly Lys1 5 1047815PRTArtificial SequenceSynthetic Peptide 478Leu Ile Lys Asn Asn Ala Ser Thr Asp Tyr Asp Leu Ser Asp Lys1 5 10 154799PRTArtificial SequenceSynthetic Peptide 479Ile Leu Lys Ser Pro Glu Ile Gln Arg1 54809PRTArtificial SequenceSynthetic Peptide 480Leu Asn Pro Tyr Ala Lys Thr Met Arg1 548115PRTArtificial SequenceSynthetic Peptide 481Lys Leu Glu Ala Ala Ala Thr Ala Leu Ala Thr Lys Ser Glu Lys1 5 10 1548221PRTArtificial SequenceSynthetic Peptide 482Thr Ile Leu Ser Asn Gln Thr Val Asp Ile Pro Glu Asn Val Glu Ile1 5 10 15Thr Leu Lys Gly Arg 2048322PRTArtificial SequenceSynthetic Peptide 483Tyr Val Ala Ser Tyr Leu Leu Ala Ala Leu Gly Gly Asn Ser Ser Pro1 5 10 15Ser Ala Lys Asp Ile Lys 2048417PRTArtificial SequenceSynthetic Peptide 484Leu Lys Thr Glu Gly Ser Asp Leu Cys Asp Arg Val Ser Glu Met Gln1 5 10 15Lys48510PRTArtificial SequenceSynthetic Peptide 485Ser Ala Val Pro Pro Gly Ala Asp Lys Lys1 5 1048611PRTArtificial SequenceSynthetic Peptide 486Arg Ser Ala Val Pro Pro Gly Ala Asp Lys Lys1 5 1048724PRTArtificial SequenceSynthetic Peptide 487Ser Ala Val Pro Pro Gly Ala Asp Lys Lys Ala Glu Ala Gly Ala Gly1 5 10 15Ser Ala Thr Glu Phe Gln Phe Arg 2048824PRTArtificial SequenceSynthetic Peptide 488Ser Ala Val Pro Pro Gly Ala Asp Lys Lys Ala Glu Ala Gly Ala Gly1 5 10 15Ser Ala Thr Glu Phe Gln Phe Arg 2048911PRTArtificial SequenceSynthetic Peptide 489Arg Ser Ala Val Pro Pro Gly Ala Asp Lys Lys1 5 1049024PRTArtificial SequenceSynthetic Peptide 490Ser Ala Val Pro Pro Gly Ala Asp Lys Lys Ala Glu Ala Gly Ala Gly1 5 10 15Ser Ala Thr Glu Phe Gln Phe Arg 2049111PRTArtificial SequenceSynthetic Peptide 491Asp Val Ile Glu Glu Tyr Phe Lys Cys Lys Lys1 5 104929PRTArtificial SequenceSynthetic Peptide 492Val Ile Ile Glu Lys Tyr Tyr Thr Arg1 549310PRTArtificial SequenceSynthetic Peptide 493Ile Gly Lys Pro His Thr Val Pro Cys Lys1 5 1049411PRTArtificial SequenceSynthetic Peptide 494Ala Glu Asp Lys Glu Trp Ile Pro Val Thr Lys1 5

1049511PRTArtificial SequenceSynthetic Peptide 495Ser Leu Glu Lys Val Cys Ala Asp Leu Ile Arg1 5 1049615PRTArtificial SequenceSynthetic Peptide 496Phe Asn Gly Gln Phe Lys Thr Tyr Gly Ile Cys Gly Ala Ile Arg1 5 10 1549714PRTArtificial SequenceSynthetic Peptide 497Asn Thr Lys Gly Gly Asp Ala Pro Ala Ala Gly Glu Asp Ala1 5 1049826PRTArtificial SequenceSynthetic Peptide 498Lys Pro Leu Pro Asp His Val Ser Ile Val Glu Pro Lys Asp Glu Ile1 5 10 15Leu Pro Thr Thr Pro Ile Ser Glu Gln Lys 20 2549930PRTArtificial SequenceSynthetic Peptide 499Ile Gly Pro Lys Lys Pro Leu Pro Asp His Val Ser Ile Val Glu Pro1 5 10 15Lys Asp Glu Ile Leu Pro Thr Thr Pro Ile Ser Glu Gln Lys 20 25 3050016PRTArtificial SequenceSynthetic Peptide 500Gly Gly Lys Pro Glu Pro Pro Ala Met Pro Gln Pro Val Pro Thr Ala1 5 10 155019PRTArtificial SequenceSynthetic Peptide 501Asn Ile Gly Lys Thr Leu Val Thr Arg1 550230PRTArtificial SequenceSynthetic Peptide 502Ile Val Lys Pro Asn Gly Glu Lys Pro Asp Glu Phe Glu Ser Gly Ile1 5 10 15Ser Gln Ala Leu Leu Glu Leu Glu Met Asn Ser Asp Leu Lys 20 25 3050330PRTArtificial SequenceSynthetic Peptide 503Ile Val Lys Pro Asn Gly Glu Lys Pro Asp Glu Phe Glu Ser Gly Ile1 5 10 15Ser Gln Ala Leu Leu Glu Leu Glu Met Asn Ser Asp Leu Lys 20 25 3050430PRTArtificial SequenceSynthetic Peptide 504Ile Val Lys Pro Asn Gly Glu Lys Pro Asp Glu Phe Glu Ser Gly Ile1 5 10 15Ser Gln Ala Leu Leu Glu Leu Glu Met Asn Ser Asp Leu Lys 20 25 3050522PRTArtificial SequenceSynthetic Peptide 505Lys Val Ala Lys Val Glu Pro Ala Val Ser Ser Ile Val Asn Ser Ile1 5 10 15Gln Val Leu Ala Ser Lys 2050627PRTArtificial SequenceSynthetic Peptide 506Val Glu Pro Ala Val Ser Ser Ile Val Asn Ser Ile Gln Val Leu Ala1 5 10 15Ser Lys Ser Ala Ile Leu Glu Ala Thr Pro Lys 20 2550720PRTArtificial SequenceSynthetic Peptide 507Lys Gly Glu Gly Ala Gln Asn Gln Gly Lys Lys Gly Glu Gly Ala Gln1 5 10 15Asn Gln Ala Lys 2050820PRTArtificial SequenceSynthetic Peptide 508Lys Gly Glu Gly Ala Gln Asn Gln Ala Lys Lys Gly Glu Gly Ala Gln1 5 10 15Asn Gln Ala Lys 2050920PRTArtificial SequenceSynthetic Peptide 509Lys Gly Glu Gly Ala Gln Asn Gln Ala Lys Lys Gly Glu Gly Gly Gln1 5 10 15Asn Gln Ala Lys 2051020PRTArtificial SequenceSynthetic Peptide 510Lys Gly Glu Gly Gly Gln Asn Gln Ala Lys Lys Gly Glu Gly Ala Gln1 5 10 15Asn Gln Gly Lys 2051120PRTArtificial SequenceSynthetic Peptide 511Lys Gly Glu Gly Ala Gln Asn Gln Gly Lys Lys Gly Glu Gly Ala Gln1 5 10 15Asn Gln Gly Lys 2051220PRTArtificial SequenceSynthetic Peptide 512Lys Gly Glu Gly Ala Gln Asn Gln Ala Lys Lys Gly Glu Gly Val Gln1 5 10 15Asn Gln Ala Lys 2051319PRTArtificial SequenceSynthetic Peptide 513Ile Glu Gly Ala Gln Asn Gln Gly Lys Lys Pro Glu Gly Thr Ser Asn1 5 10 15Gln Gly Lys51420PRTArtificial SequenceSynthetic Peptide 514Lys Ile Glu Gly Ala Gln Asn Gln Gly Lys Lys Pro Glu Gly Thr Ser1 5 10 15Asn Gln Gly Lys 2051520PRTArtificial SequenceSynthetic Peptide 515Lys Gly Glu Gly Pro Gln Asn Gln Ala Lys Lys Gly Glu Gly Ala Gln1 5 10 15Asn Gln Gly Lys 2051619PRTArtificial SequenceSynthetic Peptide 516Thr Asp Thr Val Ala Asn Gln Gly Thr Lys Gln Glu Gly Val Ser Asn1 5 10 15Gln Val Lys51720PRTArtificial SequenceSynthetic Peptide 517Lys Thr Asp Thr Val Ala Asn Gln Gly Thr Lys Gln Glu Gly Val Ser1 5 10 15Asn Gln Val Lys 2051817PRTArtificial SequenceSynthetic Peptide 518Ala Ser Met Val Gln Ser Gln Glu Ala Pro Lys Gln Asp Ala Pro Ala1 5 10 15Lys51917PRTArtificial SequenceSynthetic Peptide 519Ala Ser Met Val Gln Ser Gln Glu Ala Pro Lys Gln Asp Ala Pro Ala1 5 10 15Lys52013PRTArtificial SequenceSynthetic Peptide 520Glu Met Tyr Ser Ala Ser Lys Pro Leu Lys Gly Ala Arg1 5 1052123PRTArtificial SequenceSynthetic Peptide 521Gly Ile Ser Glu Glu Thr Thr Thr Gly Val His Asn Leu Tyr Lys Met1 5 10 15Met Ser Asn Gly Ile Leu Lys 2052223PRTArtificial SequenceSynthetic Peptide 522Gly Ile Ser Glu Glu Thr Thr Thr Gly Val His Asn Leu Tyr Lys Met1 5 10 15Met Ser Asn Gly Ile Leu Lys 2052323PRTArtificial SequenceSynthetic Peptide 523Gly Ile Ser Glu Glu Thr Thr Thr Gly Val His Asn Leu Tyr Lys Met1 5 10 15Met Ser Asn Gly Ile Leu Lys 2052435PRTArtificial SequenceSynthetic Peptide 524Gly Ile Ser Glu Glu Thr Thr Thr Gly Val His Asn Leu Tyr Lys Met1 5 10 15Met Ser Asn Gly Ile Leu Lys Val Pro Ala Ile Asn Val Asn Asp Ser 20 25 30Val Thr Lys 3552535PRTArtificial SequenceSynthetic Peptide 525Gly Ile Ser Glu Glu Thr Thr Thr Gly Val His Asn Leu Tyr Lys Met1 5 10 15Met Ser Asn Gly Ile Leu Lys Val Pro Ala Ile Asn Val Asn Asp Ser 20 25 30Val Thr Lys 3552635PRTArtificial SequenceSynthetic Peptide 526Gly Ile Ser Glu Glu Thr Thr Thr Gly Val His Asn Leu Tyr Lys Met1 5 10 15Met Ser Asn Gly Ile Leu Lys Val Pro Ala Ile Asn Val Asn Asp Ser 20 25 30Val Thr Lys 3552723PRTArtificial SequenceSynthetic Peptide 527Gly Ile Ser Glu Glu Thr Thr Thr Gly Val His Asn Leu Tyr Lys Met1 5 10 15Met Ser Asn Gly Ile Leu Lys 2052835PRTArtificial SequenceSynthetic Peptide 528Gly Ile Ser Glu Glu Thr Thr Thr Gly Val His Asn Leu Tyr Lys Met1 5 10 15Met Ser Asn Gly Ile Leu Lys Val Pro Ala Ile Asn Val Asn Asp Ser 20 25 30Val Thr Lys 3552914PRTArtificial SequenceSynthetic Peptide 529Val Pro Ala Ile Asn Val Asn Asp Ser Val Thr Lys Ser Lys1 5 1053010PRTArtificial SequenceSynthetic Peptide 530Ser Lys Phe Asp Asn Leu Tyr Gly Cys Arg1 5 1053110PRTArtificial SequenceSynthetic Peptide 531Met Leu Glu Leu Val Ala Gln Lys Gln Arg1 5 1053214PRTArtificial SequenceSynthetic Peptide 532Val Gly Asp Leu Ser Pro Lys Gln Glu Glu Ala Leu Ala Lys1 5 1053315PRTArtificial SequenceSynthetic Peptide 533Asp Gln Val Lys Gln Gln Tyr Glu His Thr Val Gln Val Ser Arg1 5 10 1553418PRTArtificial SequenceSynthetic Peptide 534Ala Gln Gly Lys Pro Val Ser Gly Gln Glu Ser Ser Gln Ser Pro Tyr1 5 10 15Glu Arg53511PRTArtificial SequenceSynthetic Peptide 535Gln Tyr Asp Ile Ser Asn Pro Gln Lys Pro Arg1 5 1053632PRTArtificial SequenceSynthetic Peptide 536Asn Thr Ser Leu Thr Asp Ile Gln Asp Leu Ser Ser Ile Thr Tyr Asp1 5 10 15Gln Asp Gly Tyr Phe Lys Glu Thr Ser Tyr Lys Thr Pro Lys Leu Lys 20 25 3053714PRTArtificial SequenceSynthetic Peptide 537Ala Ile Gly Ile Asp Pro Gly Tyr Ser Lys Ala Tyr Gly Arg1 5 1053819PRTArtificial SequenceSynthetic Peptide 538Lys Val Pro Pro Ala Asp Leu Lys Met Met Cys Leu Glu Glu Asp Ala1 5 10 15Ser Glu Arg53916PRTArtificial SequenceSynthetic Peptide 539Arg Gln Pro Pro Val Ser Gln Gly Leu Leu Glu Thr Leu Lys Ala Arg1 5 10 1554010PRTArtificial SequenceSynthetic Peptide 540Leu Lys Asp Ala Val Ile Gln Asn Thr Arg1 5 1054112PRTArtificial SequenceSynthetic Peptide 541Val Gly Met Ala Ala Val Lys Leu Ala Pro Gly Lys1 5 1054223PRTArtificial SequenceSynthetic Peptide 542Glu Gly Phe Asp Val Gly Ile Ile Ala Asp Pro Leu Tyr Ile Leu Asp1 5 10 15Asn Lys Ala Gln Thr Phe Arg 2054316PRTArtificial SequenceSynthetic Peptide 543Thr Glu Asp Thr Gln His Cys Gly Glu Gly Lys Gly Phe Leu Gln Lys1 5 10 1554419PRTArtificial SequenceSynthetic Peptide 544Gly Phe Leu Gln Lys Ser Pro Ser Lys Glu Pro His Phe Thr Asp Phe1 5 10 15Glu Gly Lys54514PRTArtificial SequenceSynthetic Peptide 545Ser Pro Ser Lys Glu Pro His Phe Thr Asp Phe Glu Gly Lys1 5 1054611PRTArtificial SequenceSynthetic Peptide 546Ala Ala Leu Lys Val Glu Glu Ser Glu Leu Lys1 5 1054713PRTArtificial SequenceSynthetic Peptide 547Leu Thr Glu Lys Glu Ser Glu Cys Thr Asp Val Cys Arg1 5 1054830PRTArtificial SequenceSynthetic Peptide 548Lys Phe Thr Asp Glu Gly Asn Pro Glu Pro Val Asn Asn Asn Gly Tyr1 5 10 15Ser Cys Val Pro Ser Asp Glu Lys Asn Ser Glu Thr Pro Leu 20 25 305498PRTArtificial SequenceSynthetic Peptide 549Ser Ser Leu Thr Thr Ile Glu Lys1 555011PRTArtificial SequenceSynthetic Peptide 550Thr Thr Val Lys Ser Ser Glu Leu Gln Gln Leu1 5 1055121PRTArtificial SequenceSynthetic Peptide 551Gln Asp Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly1 5 10 15Asn Ala Gly Ser Arg 2055218PRTArtificial SequenceSynthetic Peptide 552Ser Ala Gly Gly Asp Glu Asn Lys Glu Asn Glu Arg Pro Ser Ala Gly1 5 10 15Ser Lys55313PRTArtificial SequenceSynthetic Peptide 553Tyr Gln Ser Asn Pro Lys Val Met Asn Leu Ile Ser Lys1 5 1055416PRTArtificial SequenceSynthetic Peptide 554Glu His Tyr Asp Ser Leu Thr Glu Leu Val Asp Tyr Leu Lys Gly Lys1 5 10 1555525PRTArtificial SequenceSynthetic Peptide 555Ile Pro Phe Leu Glu Phe Ser Cys Pro Gly Val Pro Pro Gly Leu Glu1 5 10 15Thr Leu Lys Glu Thr Pro Ala Pro Arg 20 2555620PRTArtificial SequenceSynthetic Peptide 556Ser Pro Trp Ile Glu Thr Asp Ile Gly Tyr Ser Ala Leu Ile Asn Lys1 5 10 15Glu Gly Pro Arg 2055737PRTArtificial SequenceSynthetic Peptide 557Met Ala Ser Pro Glu Pro Leu Arg Gly Gly Asp Gly Ala Arg Ala Ser1 5 10 15Arg Glu Pro His Thr Glu Ala Ser Phe Pro Leu Gln Glu Ser Glu Ser 20 25 30Pro Lys Glu Ala Lys 3555828PRTArtificial SequenceSynthetic Peptide 558Gln Ser Ser Leu Thr Met Asp Gly Gly Asp Val Pro Leu Leu Glu Asp1 5 10 15Met Ala Ser Gly Ile Val Glu Leu Phe Gln Lys Lys 20 2555920PRTArtificial SequenceSynthetic Peptide 559Ala Leu Ala Gly Cys Asp Phe Leu Thr Ile Ser Pro Lys Leu Leu Gly1 5 10 15Glu Leu Leu Lys 2056011PRTArtificial SequenceSynthetic Peptide 560Leu Leu Gly Glu Leu Leu Lys Asp Asn Ser Lys1 5 1056123PRTArtificial SequenceSynthetic Peptide 561Leu Ala Pro Ala Leu Ser Val Lys Ala Ala Gln Thr Ser Asp Ser Glu1 5 10 15Lys Ile His Leu Asp Glu Lys 2056213PRTArtificial SequenceSynthetic Peptide 562Glu Leu Ser Ala Thr Ser Ser Thr Gln Lys Ile Thr Lys1 5 1056318PRTArtificial SequenceSynthetic Peptide 563Gly Ile Thr Gly Ile Glu Asp Lys Glu Ala Trp His Gly Lys Pro Leu1 5 10 15Pro Lys56418PRTArtificial SequenceSynthetic Peptide 564Asn Met Ala Glu Gln Ile Ile Gln Glu Ile Tyr Ser Gln Val Gln Ser1 5 10 15Lys Lys56523PRTArtificial SequenceSynthetic Peptide 565Ser Gln Thr Glu Glu His Glu Glu Ala Gly Pro Leu Pro Thr Lys Val1 5 10 15Asn Leu Ala His Ser Glu Ile 2056627PRTArtificial SequenceSynthetic Peptide 566Ile Ala Trp Thr His Ile Thr Ile Pro Glu Ser Leu Lys Gln Gly Gln1 5 10 15Val Glu Asp Glu Trp Tyr Ser Leu Ser Gly Arg 20 2556736PRTArtificial SequenceSynthetic Peptide 567Asn Gln Leu Glu Lys Tyr Ile Ser Glu Arg Thr Ser Gln Asp Ser Asn1 5 10 15Tyr Gly Gly Lys Ile Pro Ile Val Cys Phe Ala Gln Gly Gly Gly Arg 20 25 30Glu Thr Leu Lys 3556813PRTArtificial SequenceSynthetic Peptide 568Asn Glu Val Gly Glu Arg His Gly His Gly Lys Ala Arg1 5 1056928PRTArtificial SequenceSynthetic Peptide 569Thr Leu Lys Leu Thr Thr Pro Thr Tyr Gly Asp Leu Asn His Leu Val1 5 10 15Ser Ala Thr Met Ser Gly Val Thr Thr Cys Leu Arg 20 2557016PRTArtificial SequenceSynthetic Peptide 570Leu Tyr Ser Met Lys Phe Gln Gly Pro Asp Asn Gly Gln Gly Pro Lys1 5 10 1557135PRTArtificial SequenceSynthetic Peptide 571Lys Pro Leu Leu Glu Ser Gly Thr Leu Gly Thr Lys Gly Asn Val Gln1 5 10 15Val Val Ile Pro Phe Leu Thr Glu Ser Tyr Ser Ser Ser Gln Asp Pro 20 25 30Pro Glu Lys 3557231PRTArtificial SequenceSynthetic Peptide 572Gly Asn Val Gln Val Val Ile Pro Phe Leu Thr Glu Ser Tyr Ser Ser1 5 10 15Ser Gln Asp Pro Pro Glu Lys Ser Ile Pro Ile Cys Thr Leu Lys 20 25 3057322PRTArtificial SequenceSynthetic Peptide 573Ser Ile Pro Ile Cys Thr Leu Lys Asn Phe Pro Asn Ala Ile Glu His1 5 10 15Thr Leu Gln Trp Ala Arg 2057410PRTArtificial SequenceSynthetic Peptide 574Gly Ile Lys Leu Val Val Ala Asp Thr Arg1 5 1057511PRTArtificial SequenceSynthetic Peptide 575Ile Tyr Lys Thr Asp Arg Asp Lys Tyr Asn Arg1 5 1057611PRTArtificial SequenceSynthetic Peptide 576Ile Tyr His Pro Asn Val Asp Lys Leu Gly Arg1 5 1057717PRTArtificial SequenceSynthetic Peptide 577Ile Cys Leu Asp Ile Leu Lys Asp Lys Trp Ser Pro Ala Leu Gln Ile1 5 10 15Arg57817PRTArtificial SequenceSynthetic Peptide 578Ile Cys Leu Asp Ile Leu Lys Asp Lys Trp Ser Pro Ala Leu Gln Ile1 5 10 15Arg57919PRTArtificial SequenceSynthetic Peptide 579Lys Ser Glu Asp Asp Gly Ile Gly Lys Glu Asn Leu Ala Ile Leu Glu1 5 10 15Lys Ile Lys5809PRTArtificial SequenceSynthetic Peptide 580Val Asp Glu Asn Gly Lys Ile Ser Arg1 558112PRTArtificial SequenceSynthetic Peptide 581Tyr Tyr Lys Val Asp Glu Asn Gly Lys Ile Ser Arg1 5 1058213PRTArtificial SequenceSynthetic Peptide 582Cys Cys Leu Thr Tyr Cys Phe Asn Lys Pro Glu Asp Lys1 5 1058323PRTArtificial SequenceSynthetic Peptide 583Glu Lys Glu Glu Phe Ala Val Pro Glu Asn Ser Ser Val Gln Gln Phe1 5 10 15Lys Glu Glu Ile Ser Lys Arg 2058410PRTArtificial SequenceSynthetic Peptide 584Val Lys Ile Tyr Thr Phe Asn Gln Ser Arg1 5 1058512PRTArtificial SequenceSynthetic Peptide 585Ala His Val Tyr Val Glu Glu Val Pro Trp Lys Arg1 5 1058622PRTArtificial SequenceSynthetic Peptide 586Asp Ile Val Leu Gln Lys Phe Ala Gly Pro Tyr Asp Lys Gly Glu Tyr1 5 10 15Ser Pro Ser Val Gln Lys 2058723PRTArtificial SequenceSynthetic Peptide 587Ser Arg Pro Gly Ser Val Val Pro Ala Asn Leu Phe Gln Gly Ile Lys1 5 10 15Thr Val Asn Pro Thr Phe Arg 2058826PRTArtificial SequenceSynthetic Peptide 588Tyr Val Asp Lys Leu Glu Lys Ile Phe Gln Asn Ala Pro Thr Asp Pro1 5 10 15Thr Gln Asp Phe Ser Thr Gln Val Ala Lys

20 2558926PRTArtificial SequenceSynthetic Peptide 589Tyr Val Asp Lys Leu Glu Lys Ile Phe Gln Asn Ala Pro Thr Asp Pro1 5 10 15Thr Gln Asp Phe Ser Thr Gln Val Ala Lys 20 2559027PRTArtificial SequenceSynthetic Peptide 590Lys Tyr Val Asp Lys Leu Glu Lys Ile Phe Gln Asn Ala Pro Thr Asp1 5 10 15Pro Thr Gln Asp Phe Ser Thr Gln Val Ala Lys 20 2559114PRTArtificial SequenceSynthetic Peptide 591Phe Ala Ser Phe Pro Asp Tyr Leu Val Ile Gln Ile Lys Lys1 5 1059217PRTArtificial SequenceSynthetic Peptide 592Ser Gly Lys Pro Pro Ile Ser Asp Met Phe Ser Asp Leu Lys Asp Gly1 5 10 15Arg59311PRTArtificial SequenceSynthetic Peptide 593His Phe Cys Asn Lys Leu Trp Asn Ala Thr Lys1 5 1059418PRTArtificial SequenceSynthetic Peptide 594Leu Gly Asp Val Ile Ser Ile Gln Pro Cys Pro Asp Val Lys Tyr Gly1 5 10 15Lys Arg59515PRTArtificial SequenceSynthetic Peptide 595Ile Val Ser Gln Leu Leu Thr Leu Met Asp Gly Leu Lys Gln Arg1 5 10 1559618PRTArtificial SequenceSynthetic Peptide 596Glu Leu Gln Glu Leu Val Gln Tyr Pro Val Glu His Pro Asp Lys Phe1 5 10 15Leu Lys59715PRTArtificial SequenceSynthetic Peptide 597Lys Ser Pro Val Ala Lys Asp Val Asp Leu Glu Phe Leu Ala Lys1 5 10 1559817PRTArtificial SequenceSynthetic Peptide 598Asn Val Asn Ala Gly Gly His Lys Leu Gly Leu Gly Leu Glu Phe Gln1 5 10 15Ala59913PRTArtificial SequenceSynthetic Peptide 599Ile Glu Gly Asp Pro Gln Gly Val Gln Gln Ala Lys Arg1 5 1060024PRTArtificial SequenceSynthetic Peptide 600Ile His Val Lys Ser Gly Asp His Met Lys Gly Ala Arg Met Leu Ile1 5 10 15Arg Val Ala Asn Asn Ile Ser Lys 2060129PRTArtificial SequenceSynthetic Peptide 601Asn Tyr Gln Gln Asn Tyr Gln Asn Ser Glu Ser Gly Glu Lys Asn Glu1 5 10 15Gly Ser Glu Ser Ala Pro Glu Gly Gln Ala Gln Gln Arg 20 2560235PRTArtificial SequenceSynthetic Peptide 602Glu Val Lys Glu Asp Asp Lys Ala Pro Gly Glu Leu Glu Glu Gln Leu1 5 10 15Ser Glu Asp Gly Ser Ala Pro Glu Lys Gly Glu Val Lys Gly Asn Ala 20 25 30Ser Leu Arg 35

Claims

1. A method for determining the presence of at least one ubiquitinated polypeptide in a biological sample comprising: a. Contacting the sample with at least one hydrolyzing agent, wherein the hydrolyzing agent is capable of cleaving a ubiquitinated polypeptide to produce at least one ubiquitin remnant peptide, to obtain a hydrolyzed sample; b. Contacting the hydrolyzed sample with a substrate comprising at least one immobilized binding partner; wherein the at least one immobilized binding partner preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant; c. Removing the hydrolyzed sample from the substrate in a manner such that the at least one ubiquitin remnant peptide would remain bound to the immobilized binding partner; d. Contacting the substrate with an elution solution, where in the least one ubiquitin remnant peptide would dissociate from the immobilized binding partner into the elution solution; e. Determining the presence of a least one ubiquitinated polypeptide in the biological sample when the elution solution contains the at least one least ubiquitin remnant peptide.

2. The method of claim 1, wherein the biological sample is derived from saliva, mucous, tears, blood, serum, lymph fluids, buccal cells, mucosal cells, biopsy tissue, cerebrospinal fluid, semen, feces, plasma, urine, a suspension of cells, or a suspension of cells and viruses.

3. The method of claim 1, wherein the hydrolyzing agent is an enzyme selected from the group consisting of trypsin, Lysine-C endopeptidase (LysC), arginine-C endopeptidase (ArgC), Asp-N, glutamic acid endopeptidase (GluC) and chymotrypsin; or combinations thereof

4. The method of claim 1, wherein the substrate comprises a gel matrix.

5. The method of claim 1, wherein the substrate comprises polymer beads.

6. The method of claim 1, wherein the at least one immobilized binding partner comprises an antibody or ubiquitin remnant peptide binding fragment thereof.

7. The method of claim 6, wherein the antibody is a polyclonal antibody.

8. The method of claim 6, wherein the antibody is a monoclonal antibody.

9. The method of claim 1 wherein a portion of the elution solution is analyzed by mass spectrometry.

10. The method of claim 1, wherein step (e) is performed by tandem MS.

11. The method of claim 1, wherein the amino acid sequence of at least one ubiquitin remnant peptide present in the elution solution, is determined.

12. The method of claim 11, wherein the sequence is compared to the sequence of the ubiquitinated polypeptide to determine the site of ubiquitination.

13. The method of claim 1, wherein the elution solution further comprises at least one standard peptide, wherein the at least one standard peptide has the substantially the same amino acid sequence as the at least one distinct peptide but a different measured accurate mass.

14. The method of claim 1 wherein ubiquitinated polypeptide comprises a ubiquitin-like protein.

15. The method of claim 1 the ubiquitin-like protein is selected from the group consisting of NEDD8 and ISG15.

16. The method of claim 1, wherein the hydrolyzing agent is trypsin.

17. The method of claim 1, wherein the biological sample is depleted of a protein selected from the group consisting of albumin, IgG, IgA, transferrin, haptoglobin, and anti-trypsin; or combinations thereof, prior to step (a).

18. An isolated antibody that preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant, wherein the antibody comprises a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

19. A method for determining whether a patient is has or is likely to have or develop a disease associated with a least one ubiquitinated polypeptide comprising: a. Obtaining a biological sample from the patient; b. Contacting the sample with at least one hydrolyzing agent, wherein the hydrolyzing agent is capable of cleaving the ubiquitinated polypeptide to produce at least one ubiquitin remnant peptide, to obtain a hydrolyzed sample; c. Contacting the hydrolyzed sample with a substrate comprising at least one immobilized binding partner; wherein the at least one immobilized binding partner preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant; d. Removing the hydrolyzed sample from the substrate in a manner such that the at least one ubiquitin remnant peptide would remain bound to the immobilized binding partner; e. Contacting the substrate with an elution solution, where in the least one ubiquitin remnant peptide would dissociate from the immobilized binding partner into the elution solution; and f. Determining the presence of a least one ubiquitinated polypeptide in the biological sample when the elution solution contains the at least one least ubiquitin remnant peptide; g. Determining that the patient is has or is likely to have or develop a disease associated with a least one ubiquitinated polypeptide if the least one ubiquitinated polypeptide is present in the biological sample.

20. A method for determining whether a disease is associated with at least one ubiquitinated polypeptide comprising: a. Obtaining a biological sample from a patient having the disease; b. Contacting the sample with at least one hydrolyzing agent, wherein the hydrolyzing agent is capable of cleaving a ubiquitinated polypeptide to produce at least one ubiquitin remnant peptide, to obtain a hydrolyzed sample; c. Contacting the hydrolyzed sample with a substrate comprising an at least one immobilized binding partner; wherein the at least one immobilized binding partner preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant; d. Removing the hydrolyzed sample from the substrate in a manner such that the at least one ubiquitin remnant peptide would remain bound to the immobilized binding partner; e. Contacting the substrate with an elution solution, where in the least one ubiquitin remnant peptide would dissociate from the immobilized binding partner into the elution solution; and f. Determining the presence of a least one ubiquitinated polypeptide in the biological sample when the elution solution contains the at least one least ubiquitin remnant peptide; g. Determining that the disease is associated with the presence of the at least one ubiquitinated polypeptide if the least one ubiquitinated polypeptide is absent in the biological sample of a healthy individual.

Images