US20110111424A1 - Analysis of ubiquitinated polypeptides

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US20110111424A1

Publication number: US20110111424A1
Application number: US12/967,284
Authority: US
Inventors: II John Edward Rush; Jing Li; Ailan Guo
Original assignee: Cell Signaling Technology Inc
Current assignee: Cell Signaling Technology Inc
Priority date: 2001-06-21
Filing date: 2010-12-14
Publication date: 2011-05-12
Also published as: US9181326B2; US20130245237A1

The invention relates to 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.

RELATED APPLICATIONS

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

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

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.
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.
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.
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.
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).
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).
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).
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).
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.
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

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

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:

FIG. 1 depicts a cartoon of the formation of a ubiquitin remnant

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

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).
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.
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.
Prior to describing various embodiments of the current invention, the following definitions are provided:
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.
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).
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.
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.
The invention also encompasses the novel ubiquitin remnant peptides disclosed herein in Table 4 as well as fragments and variants thereof.
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.
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.times.100%). In one embodiment, the two sequences are the same length.
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.
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.
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.
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.
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.
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.
Fragment ions in the MS/MS and MS³spectra 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.
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.
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.
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.
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%).
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.
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.
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.
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.
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.
The isolated antibodies and/or isolated binding partners of the invention can be used in the methods of the invention.
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.
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.
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).
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.
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).
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.
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.
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, IgA₁and 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.
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.
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.
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.
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.
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).
As one non-limiting example, the following method can be used to identify the CDRs of an antibody.
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^thresidue 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.
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.
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).
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.
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).
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).
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.
In further embodiments, the anti-ubiquitin remnant peptide antibodies of the invention have a dissociation constant (K_D) of 10⁻⁷M 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 10⁻⁹M or less for a ubiquitin remnant peptide.
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.
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′)₂fragments, 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′)₂fragments, 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.
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.
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.)
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.
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.
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).
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.
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.
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.
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.)
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.
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).
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.
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.
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.
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).
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).
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.
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.
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).
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.
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.
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.
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.
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.
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.
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

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


	Monoclonal anti-Ubiquitin	Polyclonal anti-Ubiquitin
	Remnant Antibodies	Remnant Antibodies

		BL3415
		BL3416
	D4A7A10	BL4933
		BL4934
	D28B6G9	BL4935
		BL4936

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:

	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)

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

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 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)
	AKIQDKEGIPPDQQR (SEQ ID NO: 14)

33	IQDK*EGIPPDQQR (SEQ ID NO: 15)
	AKIQDK*EGIPPDQQR (SEQ ID NO: 16)
	AKIQDKEGIPPDQQR (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)

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 C₁₈columns 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.
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

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.

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

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 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,	AKIQDKEGIPPDQQR	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	TVVGKEEEGSDPEKALR	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	THGRAKSYKCGECGK	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,	RTTHGFKMSKIYK*	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	AKDM#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	KKTSEEILQHLQNIVDFGKNVMK*	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	KESDLSGAQIKLR	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,	MQANNAKAVSARTEAIKALVK	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	SAVPPGADKKAEAGAGSATEFQFR	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	NQLEKYISERTSQDSNYGGKIPIV	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,	KSEDDGIGKENLAILEKIK*	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,	EVKEDDKAPGELEEQLSEDGSAP	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

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.
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).
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).
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).
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.
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.

Cell/Tissue Type

Treatment 1

Treatment 2

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;

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.