# Patent

## Title
Analysis of ubiquitinated polypeptides

## Abstract

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.

## Background
RELATED APPLICATIONS
This Application is a divisional of U.S. Ser. No. 12/967,824 filed Dec. 14, 2010, which claims benefit from U.S. provisional patent application Ser. No. 61/286,486 filed Dec. 15, 2009, the entire disclosures of which are hereby incorporated by reference. U.S. Ser. No. 12/967,824, filed Dec. 14, 2010 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 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, 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/NIS 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 8-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 ubiquitinproteosome 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), NFKB, cystic fibrosis transduction receptor, p53, ornithine decarboxylase (ODC), 7-membrane spanning receptors, Cdc25 (phosphotyrosme phosphatase), Rb, Ga, 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 a-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 ubiquinated 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 (scFvs), 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 8-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 antiubiquitin 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 MS3 spectra are generally highly specific and diagnostic for peptides of interest. In contrast, to prior art methods, the identification of peptide diagnostic signatures provides for a way to perform highly selective analysis of a complex protein mixture, such as a cellular lysate in which there may be greater than about 100, about 1000, about 10,000, or even about 100,000 different kinds of proteins. Thus, while conventional mass spectroscopy would not be able to distinguish between peptides with different sequences but similar m/z ratios (which would tend to co-elute with any labeled standard being analyzed), the use of peptide fragmentation methods and multistage mass spectrometry in conjunction with LC methods, provide a way to detect and quantify target proteins which are only a small fraction of a complex mixture (e.g., present in less than 2000 copies per cell or less than about 0.001% of total cellular protein) through these diagnostic signatures.
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, subiquitin 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, IgA1 and IgA2) or subclass of immunoglobulin molecule. Preferably, an antibody of the invention comprises, or alternatively consists of, a VH domain, VH CDR, VL domain, or VL CDR having an amino acid sequence of any one of the antibodies listed in Table 1, or a fragment or variant thereof. In a preferred embodiment, the immunoglobulin is an IgG1 isotype. In another preferred embodiment, the immunoglobulin is an IgG4 isotype. Immunoglobulins may have both a heavy and light chain. An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa or lambda forms. Antibodies of the invention may also include multimeric forms of antibodies. For example, antibodies of the invention may take the form of antibody dimers, trimers, or higher-order multimers of monomeric immunoglobulin molecules. Dimers of whole immunoglobulin molecules or of F(ab′)2 fragments are tetravalent, whereas dimers of Fab fragments or scFv molecules are bivalent. Individual monomers withon an antibody multimer may be identical or different, i.e., they may be heteromeric or homomeric antibody multimers. For example, individual antibodies within a multimer may have the same or different binding specificities.
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 [Nsuccinimidyl 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 (Jan. 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 24th residue is typically a cysteine. The CDR-L1 ends on the residue before a tryptophan residue. Typically, the sequence containing the tryptophan is either Trp-Tyr-Gln, Trp-Leu-Gln Trp-Phe-Gln, or Trp-Tyr-Leu, where the last residue within the CDR-L1 domain is the residue before the TRP in all of these sequences.
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 (KD) of 10−7 M or less for a ubiquitin remnant peptide. In preferred embodiments, the anti-ubiquitin remnant peptide antibodies of the invention have a dissociation constant (KD) of 10−9 M or less for a ubiquitin remnant peptide.
In further embodiments, antibodies of the invention have an off rate (koff) of 10−3/sec or less. In preferred embodiments, antibodies of the invention have an off rate (koff) of 10−4/sec or less. In other preferred embodiments, antibodies of the invention have an off rate (koff) 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′)2 fragments, Fd fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and scFvs). The present invention further provides mixtures of the anti-ubiquitin remnant peptide antibodies wherein the mixture corresponds to one, two, three, four, five, ten, fifteen, twenty, or more different the anti-ubiquitin remnant peptide antibodies of the invention (e.g., whole antibodies, Fabs, F(ab′)2 fragments, Fd fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and scFvs)). The present invention also provides for compositions comprising, or alternatively consisting of, one, two, three, four, five, ten, fifteen, twenty, or more the anti-ubiquitin remnant peptide antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof). A composition of the invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty, or more amino acid sequences of one or more of the anti-ubiquitin remnant peptide antibodies or fragments or variants thereof. Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one or more antibodies of the invention.
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 (O), 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 GlyGly 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 eurodegenerative 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 Can 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 Spectrum. 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 P 19 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, electro fusion, 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







(SEQ ID NO: 3)









GFTISSNYYIYWV


 



CDR2







(SEQ ID NO: 4)









CIYGGSSGTTLYASWAKG


 



CDR3







(SEQ ID NO: 5)









DFRGADYSSYDRIWDTRLDL


 



Light Chain:



CDR1







(SEQ ID NO: 6)









QSSENVYNKNWLS


 



CDR2







(SEQ ID NOL: 7)









KASTLAS


 



CDR3







(SEQ ID NO: 8)









AGDYGGTGDAFV






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) AK*IQDK*EGIPPDQQR (SEQ ID NO: 14)


 


33
IQDK*EGIPPDQQR (SEQ ID NO: 15)



AKIQDK*EGIPPDQQR (SEQ ID NO:



16) AK*IQDK*EGIPPDQQR (SEQ ID NO: 17)


 


48
LIFAGK*QLEDGR (SEQ ID NO: 18)



LIFAGK*QLEDGRTLSDYNIQK (SEQ ID NO: 19)



LIFAGK*QLEDGRTLSDYNIQKESTLHLVLR



(SEQ ID NO: 20)


 


63
TLSDYNIQK*ESTLHLVLR (SEQ ID NO: 21)









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-PakC18 columns as described in Rush et al., U.S. Pat. No. 7,300,753). Following purification, peptides are lyophilized and then resuspended in MOPS buffer (50 mM MOPS/NaOH pH 7.2, 10 mM Na2HPO4, 50 mM NaC1) 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 1[11, 160 iug) 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 ddH20. Peptides were eluted twice from beads by incubation with 50 IA 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.













TABLE 3





Expt
Antibody
Cell/Tissue Type
Treatment 1
Treatment 2
Redundant
Non-Redundant



















3114
BL4936
mouse heart
447
332


3115
BL4936
mouse liver
790
591


3116
BL4936
Embryo mouse
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
Reperfusion
760
554




ischemia 30 R


3576
BL4936
rat brain
ischemia
Reperfusion
809
580




ischemia 30′ R


3577
BL4936
rat brain
ischemia
Reperfusion
741
551




ischemia 24 h R


3578
BL4936
rat brain
ischemia
Reperfusion
773
567




ischemia 24 h R


3970
BL4936
rat brain sham
untreated

693
499


3971
BL4936
rat brain
Reperfusion

829
604




ischemia 30′ R


3972
BL4936
rat brain
Reperfusion

816
620




ischemia 24 h 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
D24B6G
Jurkat
pervanadate
calyculin
137
105


5972
BL49
H3122 Silac
inhibitor

353
200


5973
BL49
H3122 Silac
inhibitor

247
185


5974
BL49
H3122 Silac
inhibitor

391
245


6090
BL49
H2228 silac
inhibitor

193
135




Dana Farber


6093
BL49
H3122 Silac
inhibitor

178
140




Dana Farber


6131
BL49
H1703
normal

978
691


6362
D24B
Jurkat
pervanad
calyculin
431
283


6586
BL49
U266
control

793
539


6587
BL49
U266
MG132

791
522


6588
BL49
U266
MG132

1074
867


6589
BL49
H929
control

1265
764


6590
BL49
H929
MG132

712
468


6591
BL49
H929
MG132

551
467


6846
BL49
H1703


735
484


6847
BL49
H1703


1143
841


6916
BL49
RAW 264.7
normal

1366
736


6917
BL49
RAW 264.7
LPS

1396
746


6918
BL49
RAW 264.7
LPS

1424
771


6919
BL49
RAW 264.7
MG132

1473
871


6939
D4A7
Jurkat
pervanad
calyculin
286
240


6941
BL49
Jurkat
pervanad
calyculin
130
102


8149
D4A7
Jurkat
calyculin
pervanad
613
445


8158
D4A7
mouse muscle
untreated

886
651


8159
D4A7
mouse spleen
untreated

1355
1033


8160
D4A7
mouse testis
untreated

1096
872


8161
D4A7
mouse thymus
untreated

827
623


8241
D4A7
LNCaP
control

940
801


8242
D4A7
LNCaP
AAG

978
826


8243
D4A7
LNCaP
AAG

561
474












8244
D4A7
LNCaP
Geldanamycin
874
747


8245
D4A7
LNCaP
Geldanamycin
665
569


8246
D4A7
LNCaP
Velcade

970


8247
D4A7
LNCaP
Velcade

1056









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 15 from Mouse Liver

















SEQ





Ubiquitinated

ID


Row
Protein Type
Protein
Residue
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*TQSIYDDKN
27



channel,


HR




transporter







or cell







surface







protein






 


7
Chromatin,
HID; H1C
%45; %46
KASGPPVSELITK*AVAASK
28



DNA-







binding,







DNA repair







or DNA







replication







protein






 


8
Chromatin,
H1D; H1E;
%63;
K*ALAAAGYDVEK
29



DNA-
H1C; H1T
%63;





binding,

%64; %66





DNA repair







or DNA







replication







protein






 


9
Chromatin,
H1D; H1E;
%63;
K*ALAAAGYDVEKNNSR
30



DNA-
H1C; H1T
%63;





binding,

%64; %66





DNA repair







or DNA







replication







protein






 


10
Chromatin,
H1D; H1E;
%74; %74;
ALAAAGYDVEK*NNSR
31



DNA-
H1C; H1T
%75; %77





binding,







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*
34



DNA-
H2A0; H2AE
%118;
KTESHH K




binding,

%119





DNA repair







or







DNA







replication







protein






 


14
Chromatin,
H2A.1;
%120; 119;
VTIAQGGVLPNIQAVLLPKK
35



DNA-
H2A0; H2AE
%120
*TESHH K




binding,







DNA repair







or







DNA







replication







protein






 


15
Chromatin,
H2A.1;
%119;
VTIAQGGVLPNIQAVLLPK* K
36



DNA-binding,
H2AX;
%118;





DNA repair or
HIST2H2AB;
19;





DNA
HIST2H2AC;
119; %118;





replication
H2A0;
%119;





protein
H2A.4;
%119;






H2AE;
%119;






H2AL;
%119






H2AFJ





 


16
Chromatin,
H2A.1;
%120;
VTIAQGGVLPNIQAVLLPKK *
37



DNA-
H2AX;
%119; 120;





binding,
HIST2H2AB;
120;





DNA repair
HIST2H2AC;
119; 120;





or DNA
H2A0;
%120;





replication
H2A.4;
%120;





protein
H2AE;
%120






H2AL;







H2AFJ





 


17
Unassigned
H2AE
%120
VTIAQGGVLPNIQAVLLPKK
38






*TESHH KPK



 


18
Chromatin,
H2AFJ
%119
VTIAQGGVLPNIQAVLLPK*
39



DNA-


KTESQK




binding,







DNA repair







or DNA







replication







protein






 


19
Chromatin,
H2AFJ
%120
VTIAQGGVLPNIQAVLLPKK
40



DNA-







binding,







DNA repair







or DNA






 


20
Chromatin,
H2AFY
%116
GVTIASGGVLPNIHPELLAK*
41



DNA-







binding,







DNA repair







or DNA






 


21
Chromatin,
H2AFY
%116
GVTIASGGVLPNIHPELLAK*
42



DNA-







binding,







DNA repair







or DNA






 


22
Chromatin,
H2AFY
%117
GVTIASGGVLPNIHPELLAKK
3



DNA-


*R




binding,







DNA repair







or DNA







replication







protein






 


23
Chromatin,
H2AL
%120
VTIAQGGVLPNIQAVLLPKK
4



DNA-


*TETHH K




binding,







DNA repair







or DNA







replication







protein






 


24
Chromatin,
H2AX
%119
K*SSATVGPK
5



DNA-







binding,







DNA repair







or DNA







replication







protein






 


25
Chromatin,
H2AX;
%118; 119
LLGGVTIAQGGVLPNIQAVL
46



DNA-
HIST2H2AB

LPK*K




binding,







DNA repair







or DNA







replication







protein






 


26
Chromatin,
H2AX;
%118; 119
NDEELNKLLGGVTIAQGGV
47



DNA-
HIST2H2AB

LPNIQA




binding,


VLLPK*K




DNA repair







or







DNA







replication







protein






 


27
Chromatin,
H2B;
%120;
AVTK*YTSSK
48



DNA-binding,
H2B1D;
%120;





DNA repair or
H2B1A;
%122;





DNA
H2B1N;
%121;





replication
H2B2E;
%121;





protein
H2B1H;
%121;






H2B1C;
%121;






Hist3h2ba
%121




 


28
Chromatin,
H2B;
%46; %46;
VLK*QVHPDTGISSK
49



DNA-
H2B1D;
%48; %47;





binding,
H2B1A;
%47; %47;





DNA repair or
H2B1N;
%47; %47;





DNA
H2B2E;
%47





replication
H2B1L;






protein
H2B1H;







H2B1C;







Hist3h2ba





 


29
Chromatin,
H2B;
%116;
HAVSEGTK*AVTK
50



DNA-binding,
H2B1D;
%116;





DNA repair or
H2B1A;
%118;





DNA
H2B1N;
%117;





replication
H2B2E;
%117;





protein
H2B1L;
%117;






H2B1H;
%117;






H2B1C;
%117;






Hist3h2ba
%117




 


30
Chromatin,
H2B1L
%121
AVTK*YTSAK
51



DNA-







binding,







DNA repair







or







DNA







replication







protein






 


31
Unassigned
HIST2H2AB;
119; 119;
VTIAQGGVLPNIQAVLLPK*
52




HIST2H2AC;
%119
KTESHK





H2A.4





 


32
Unassigned
HIST2H2AB;
125; 125;
VTIAQGGVLPNIQAVLLPKK
53




HIST2H2AC;
%125
TESHK*





H2A.4





 


33
Chaperone
HSC70;
507;
ITITNDK*GR
54




HSPA1L;
509;






HSPA2;
510;






HSP70-2;
507;






HSP70
%507




 


34
Ubiquitin
NEDD8
%48
LlYSGK*QMNDEK
55



conjugating







system






 


35
Unassigned
RPS20
%8
DTGK*TPVEPEVAIHR
56


 


36
Unknown
SPG20
%360
SSHPSEPPK*EASGTDVR
57



function






 


37
Protein
Titin
%30428
EAFSSVIIK*EPQIEPTADLTG
58



kinase,


ITNQLI




Ser/Thr


TCK




(non-







receptor)






 


38
Cytoskeletal
TUBA1B;
%370; 370;
VGINYQPPTVVPGGDLAK*V
59



protein
TUBA3D;
370; 370;
QR





TUBA4A;
370; 370;






TUBA1A;
369






TUBA1C;







TUBA8;







TUBA3C





 


39
Ubiquitin
UBA52;
11; %11
TLTGK*TITLEVEPSDTIENV K
60



conjugating
ubiquitin






system






 


40
Ubiquitin
UBA52;
6; %6; 6
MQIFVK*TLTGK
61



conjugating
ubiquitin;






system
L0C388720





 


41
Ubiquitin
UBA52;
29; %29;
AK*IQDKEGIPPDQQR
62



conjugating
ubiquitin;
29;





system
L0C388720;
106






Gm7866





 


42
Ubiquitin
UBA52;
29, 33;
AK*IQDK*EGIPPDQQR
63



conjugating
ubiquitin;
%29,





system
L0C388720;
%33; 29,






Gm7866
33;







106, 110




 


43
Ubiquitin
UBA52
33; %33;
IQDK*EGIPPDQQR
64



conjugating
ubiquitin;
33;





system
L0C388720;
110






Gm7866





 


44
Ubiquitin
UBA52;
33; %33;
AKIQDK*EGIPPDQQR
65



conjugating
ubiquitin;
33;





system
L0C388720;
110






Gm7866





 


45
Ubiquitin
UBA52;
48; %48;
LIFAGK*QLEDGR
66



conjugating
ubiquitin;
48;





system
L0C388720;
125






Gm7866





 


46
Ubiquitin
UBA52;
48; %48;
LIFAGK*QLEDGRTLSDYNI
67



conjugating
ubiquitin;
48;
QK




system
L0C388720;
125






Gm7866





 


47
Ubiquitin
UBA52;
48; %48;
LIFAGK*QLEDGRTLSDYNI
68



conjugating
ubiquitin;
48; 125
QKESTL HLVLR




system
L0C388720;







Gm7866





 


48
Ubiquitin
UBA52;
63; %63;
TLSDYNIQK*ESTLHLVLR
69



conjugating
ubiquitin;
63;





system
LOC388720;
63; 140






OTTMUSGOO







000001634;







Gm7866





 


49
Mitochondrial
1190003J15
67
CPGLLTPSQIKPGTYK*LFFD
70



protein
Rik

TER



 


50
Unassigned
1300002K09
223
SM#LEAHQAKHVK*QLLSK
71




Rik

PR



 


51
Adaptor/
14-3-3 eta;
49; 49;
NLLSVAYK*NVVGAR
72



scaffold
14-3-3
49; 50






gamma; 14-







3-3 zeta;







14-3-3 beta





 


52
Enzyme, misc.
1-Cys PRX
198
KGESVM#VVPTLSEEEAK*Q
73






CFPK



 


53
Enzyme, misc.
1-Cys PRX
198
KGESVMVVPTLSEEEAK*QC
74






FPK



 


54
Enzyme, misc.
1-Cys PRX
208
GVFTK*ELPSGK
75


 


55
Receptor,
ABCA3
503
TVVGK*EEEGSDPEK
76



channel,







transporter







or cell







surface







protein






 


56
Receptor,
ABCA3
503, 512
TVVGK*EEEGSDPEK*ALR
77



channel,







transporter







or cell







surface







protein






 


57
Receptor,
ABCA3
1620
SEGK*QDALEEFK
78



channel,







transporter







or cell







surface







protein






 


58
Unassigned
ABCB11
935
EILEK*AGQITNEALSNIR
79


 


59
Unassigned
ABCB11
935
MLTGFASQDKEILEK*AGQ
80






ITNEAL SNIR



 


60
Unassigned
ABCB11
935
M#LTGFASQDKEILEK*AGQ
81






ITNEAL 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*VONEAK
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#K1K
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*VCLIGCGFSTG
96



protein


YGSA







VK



 


76
Mitochondrial
ADH1C
186
IDGASPLDKVCLIGCGFSTG
97



protein


YGSAV







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*QWVDOSSPVLLDNPVL
103






GSMAK



 


83
Enzyme, misc.
AKR1C1
312
YISGSSFK*DHPDFPFWDEY
104


 


84
Enzyme, misc.
ALAD
87
VPK*DEQGSAADSEDSPTIE
105






AVR



 


85
Enzyme, misc.
ALAD
87
CVLIFGVPSRVPK*DEQGSA
106






ADSED 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*DLD K
110


 


90
Enzyme, misc.
ALDH1A1
434
RANNTTYGLAAGLFTK*DL
111


 


91
Enzyme, misc.
ALDH1A2
338
IFVEESIYEEFVK*
112


 


92
Unassigned
Aldh1a7
435
ANNTTYGLAAGVFTK*DLD
113


 


93
Unassigned
Aldh1a7
499
TVAMQISQK*NS
114


 


94
Enzyme, misc.
Aldh1a7;
91; 90
LLNK*LADLMERDR
115




ALDH1A





 


95
Enzyme, misc.
Aldh1a7;
91; 90
LLNK*LADLMER
116




ALDH1A





 


96
Enzyme, misc.
Aldh1a7;
255; 254
LIK*EAAGK
117




ALDH1A





 


97
Enzyme, misc.
Aldh1a7;
378; 377
WGNK*GFFVQPTVFSNVTD
118




ALDH1A





 


98
Enzyme, misc.
Aldh1a7;
378; 377
WGNK*GFFVQPTVFSNVTD
119




ALDH1A





 


99
Enzyme, misc.
Aldh1a7;
398; 397
IAK*EEIFGPVQQIMK
120




ALDH1A





 


100
Enzyme, misc.
ALDH3A2
296
LQSLLK*GQK
121


 


101
Enzyme, misc.
ALDH7A1
424
FQDEEEVFEWNNEVK*QG
122






LSSSIFT K



 


102
Enzyme, misc.
ALDOB
47
IK*VENTEENRR
123


 


103
Enzyme, misc.
ALDOB
107
GIVVGIK*LDQGGAPLAGTN
124


 


104
Enzyme, misc.
ALDOB
107
GIVVGIK*LDQGGAPLAGTN
125






KETTIQ GLDGLSER



 


105
Enzyme, misc.
ALDOB
120
LDQGGAPLAGTNK*ETTIQG
126






LDGLS 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*EQFFENYYGOSS
144



DNA-


LSLE DSQK




binding,







DNA repair







or DNA






 


124
Unknown
AUP1
250
VQQLVAK*ELGQIGTR
145



function






 


125
Unknown
BAT3
56
EH IAASVSIPSEK*QR
146



function






 


126
Unassigned
BC066028
365, 368
THGRAK*SYK*CGECGK
147


 


127
Transcriptional
BCoR-like
1491; 1464
LIVNK*NAGETLLQR
148



regulator
1; 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*PSSPHSGQDPPAP
151






PVD



 


131
Enzyme, misc.
CA3
39
D I K*H
152






DPSLQPWSASYDPGSAK



 


132
Enzyme, misc.
CA3
57
D I KH
153






DPSLQPWSASYDPGSAK*TIL







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*PPSGK
158






DWD DDQND



 


138
Chaperone
CCT-theta
538
PAGGPKPPSGK*KDWDDDQ
159


 


139
Chaperone
CCT-theta
539
VDQIIMAKPAGGPKPPSGKK
160






DWD DDQND



 


140
Unassigned
CHIC1
179
SIQK*LLEWENNR
161


 


141
Unknown
CIRH1A
642, 645,
RTTHGFK*MSK*IYK*
162



function

648




 


142
Cytoskeletal
claudin 3
216
STGPGTGTGTAYDRK*DYV
163



protein






 


143
Unassigned
CLIC4
202
LH IVKVVAK*
164


 


144
Vesicle
CLTC
629
AH IAQLCEK*AGLLQR
165


 


145
Vesicle
CLTC
1450
AVNYFSK*VK
166


 


146
Vesicle
CLTC
1452
VK*QLPLVKPYLR
167


 


147
Mitochondrial
CPS1
307
EPLFG ISTGN I ITG
168



protein


LAAGAK*SYK



 


148
Mitochondrial
CPS1
310
SYK*MSMANR
169



protein






 


149
Mitochondrial
CPS1
560
QLFSDKLNEINEK*IAPSFAVE
170



protein


SMED 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*LWDQTSSEVKEV
177






YDKNF LDSYIK



 


157
Cell cycle
CTH
47
AVVLPISLATTFK*QDFPGQS
178



regulation


SGFE 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
MEIVDHALHALTDEVIIPHS
182



scaffold


GWERE PNEDCK*PR



 


162
Adaptor/
CTNND1
517
M#EIVDHALHALTDEVIIPHS
183



scaffold


GWER EPNEDCK*PR



 


163
Adaptor/
CTNND1
710
SALRQEK*ALSAIAELLTSEH
184



scaffold


ER



 


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,
CYB5R3
240
LWYTVDK*APDAWDYSQG
190



misc.


FVNEE M#IR



 


170
Enzyme,
CYP1A1;
97; 94
IGSTPVVVLSGLNTIK*QALV R
191



misc.
CYP1A2





 


171
Enzyme,
CYP1A2
250
YLPNPALK*R
192



misc.






 


172
Enzyme,
CYP1A2
276
TVQEHYQDFNK*NSIQDITSA
193



misc.


LFK



 


173
Enzyme,
CYP1A2
294
HSENYK*DNGGLIPEEK
194



misc.






 


174
Enzyme,
CYP1A2
401
DTSLNGFHIPK*ER
195



misc.






 


175
Enzyme,
Cyp2a12
250
DSHKLEDFMIQK*VK
196



misc.






 


176
Enzyme,
Cyp2a12
252
VK*QNQSTLDPNSPR
197



misc.






 


177
Endoplasmic
CYP2A7
32
LSGK*LPPGPTPLPFVGNFLQ
198



reticulum or


LNTE QM#YNSLM#K




golgi






 


178
Endoplasmic
CYP2A7
32
LSGK*LPPGPTPLPFVGNFLQ
199



reticulum or


LNTE QMYNSLM#K




golgi






 


179
Endoplasmic
CYP2A7
32
LSGK*LPPGPTPLPFVGNFLQ
200



reticulum or


LNTE QMYNSLMK




golgi






 


180
Endoplasmic
CYP2A7;
239; 239
HLPGPQQQAFK*ELQGLEDFI
201



reticulum or
Cyp2a5

TK




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;
ELQGLEDFITK*K
205



reticulum or
Cyp2a5;
35





golgi
Cyp2a21-







ps





 


185
Enzyme,
CYP2B1;
346;
TK*MPYTDAVIHEIQR
206



misc.
Cyp2b9;
345;






Cyp2b13
345




 


186
Enzyme,
CYP2C19;
432;
KSDYFMPFSTGK*R
207



misc.
Cyp2c29;
331;






CYP2C9;
432;






Cyp2c54;
432;






Cyp2c50
432




 


187
Enzyme,
CYP2C19;
432;
SDYFMPFSTGK*R
208



misc.
Cyp2c29;
331;






CYP2C9;
432;






Cyp2c54;
432;






Cyp2c50
432




 


188
Enzyme,
CYP2C19;
84; 84
KPTVVLHGYEAVK*EALVD
209



misc.
Cyp2c50

HGEEFA GR



 


189
Unassigned
Cyp2c29
298
GTTVITSLSSVLHDSK*EFPN
210






PEM# FDPGHFLNGNGNFK



 


190
Enzyme,
Cyp2c39
399
GTTVVTSLTSVLHDSK*EFP
211



misc.


NPELF DPGHFLDANGNFK



 


191
Enzyme,
Cyp2c39;
270;
DFIDYYLIK*QK
212



misc.
Cyp2c29;
169;






CYP2C9
270




 


192
Enzyme,
Cyp2c40
375
YIDLGPNGVVHEVTCDTK*FR
213



misc.






 


193
Enzyme,
Cyp2c40;
110; 110
GK*GIGFSHGNVWK
214



misc.
L0C1000483







23





 


194
Enzyme,
Cyp2c40;
154; 154
VQEEAQWLM#K*ELKK
215



misc.
L0C1000483







23





 


195
Enzyme,
Cyp2c40;
154; 154
VQEEAQWLMK*ELKK
216



misc.
L0C1000483







23





 


196
Enzyme,
Cyp2c40;
154; 154
VQEEAQWLMK*ELK
217



misc.
L0C1000483







23





 


197
Enzyme,
Cyp2c40;
154; 154
VQEEAQWLM#K*ELK
218



misc.
L0C1000483







23





 


198
Enzyme,
Cyp2c40;
157; 157
VQEEAQWLM#KELK*
219



misc.
L0C1000483







23





 


199
Enzyme,
Cyp2c40;
157; 157
VQEEAQWLMKELK*K
220



misc.
L0C1000483







23





 


200
Enzyme,
Cyp2c40;
249; 249
IK*EHEESLDVTNPR
221



misc.
LOCI000483







23





 


201
Enzyme,
Cyp2c54
84
KPTVVLHGYEAVK*EALVD
222



misc.


HGDVF 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
226






*PLR



 


206
Enzyme,
CYP2D2
414
GTTLIPNLSSVLKDETVWEK*
227



misc.


PLR



 


207
Unassigned
Cyp2d40
252
GTTLICNLSSVLKDETVWEK
228






*PLR



 


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*GlIFNNGPTWK
235



reticulum or







golgi






 


215
Endoplasmic
CYP2E1
123
GlIFNNGPTWK*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*GQPFDPTFLIGCAPCNVI
240



reticulum or


ADILF 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*DIDLSPVTIGFGSIP R
246



reticulum or







golgi






 


226
Receptor,
CYP3A4
35
K*QGIPGPTPLPFLGTVLNYY K
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*GSIDPYLYMPFGIG
251



channel,


PR




transporter







or cell







surface







protein






 


231
Receptor,
CYP3A4
477
VMQNFSFQPCQETQIPLK*LS R
252



channel,







transporter







or cell







surface







protein






 


232
Receptor,
CYP3A43;
421; 422;
FSK*ENK
253



channel,
CYP3A5;
422; 558;





transporter
CYP3A7;
657; 422;





or cell
UVRAG;
422





surface
KIAA1802;






protein
Cyp3a44;







CYP3A4





 


233
Unassigned
Cyp3a44
422
FSK*ENKGSIDPYVYLPFGIG
254






PR



 


234
Unassigned
Cyp3a44
488
QGILOPEK*PIVLK
255


 


235
Unassigned
Cyp3a44
493
QGILOPEKPIVLK*VVPR
256


 


236
Receptor,
Cyp3a44;
116; 116;
EFGPVGIMSK*AISISKDEEW
257



channel,
CYP3A4;
16
KR




transporter
L00673748






or 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,
CYP3A5;
35; 35
K*QGIPGPKPLPFLGTVLNYY K
261



misc.
CYP3A7





 


241
Enzyme,
CYP3A5;
42; 42
QGIPGPK*PLPFLGTVLNYYK
262



misc.
CYP3A7





 


242
Receptor,
CYP3A5;
96; 96; 96
NVLVK*ECFSVFTNR
263



channel,
CYP3A7;






transporter
CYP3A4






or cell







surface







protein






 


243
Enzyme,
CYP3A5;
158.
LKEM#FPVIEQYGDILVK*YLR
264



misc.
CYP3A7;
158:






Cyp3a44
158




 


244
Enzyme,
CYP3A5;
158;
EM#FPVIEQYGDILVK*YLR
265



misc.
CYP3A7;
158:






Cyp3a44
158




 


245
Enzyme,
CYP3A5;
158;
LKEMFPVIEQYGDILVK*YLR
266



misc.
CYP3A7;
158;






Cyp3a44
158




 


246
Receptor,
CYP3A5;
143; 143;
LK*EM#FPVIEQYGDILVK
267



channel,
CYP3A7;
143; 143





transporter
Cyp3a44;






or cell
CYP3A4






surface







protein






 


247
Receptor,
CYP3A5;
143; 143;
LK*EMFPVIEQYGDILVK
268



channel,
CYP3A7;
143; 143





transporter
Cyp3a44;






or cell
CYP3A4






surface







protein






 


248
Receptor,
CYP3A5;
250; 250;
DSIEFFK*K
269



channel,
CYP3A7;
250; 250





transporter
Cyp3a44;






or cell
CYP3A4






surface







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,
CYP51A1
436
YLQDNPASGEK*FAYVPFGAGR
272



misc.






 


252
Enzyme,
CYP51A1
436
LDFNPDRYLQDNPASGEK*F
273



misc.


AYVP FGAGR



 


253
Endoplasmic
Cyp7a1
127
SIDPSDGNTTENINK*TFNK
274



reticulum or







golgi






 


254
Enzyme, misc.
CYP8B1
366
VVQEDYVLK*MASGQEYQ IR
275


 


255
Lipid
DBI
50
QATVGDVNTDRPGLLDLK*GK
276



binding







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
elF3C
860
TEPTAQQNLALQLAEK*LGS
287






LVENN ER



 


267
Translation
elF3-theta
420
EQPEK*EPELQQYVPQLQNN
288


 


268
Translation
elF3-theta
775
QSVYEEK*LKQFEER
289


 


269
Vesicle
epsin 1
107
ENMYAVQTLK*DFQYVDR
290



protein


DGKDQ GVNVR



 


270
Enzyme, misc.
esterase D
17
CFGGLQK*VFEHSSVELK
291


 


271
Lipid
FABP1
20
YQLQSQENFEPFMK*AIGLP
292



binding


EDLIQ K




protein






 


272
Lipid
FABP1
31
AIGLPEDLIQK*GK
293



binding







protein






 


273
Lipid
FABP1
36
GKDIK*GVSEIVHEGK
294



binding







protein






 


274
Lipid
FABP1
36
DIK*GVSEIVHEGK
295



binding







protein






 


275
Lipid
FABP1
46
GVSEIVHEGK*K
296



binding







protein






 


276
Lipid
FABP1
80
VK*AVVKLEGDNK
297



binding







protein






 


277
Lipid
FABP1
84
AVVK*LEGDNK
298



binding







protein






 


278
Lipid
FABP1
99
M#VTTFKGIK*
299



binding







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
FAH
186
RPMGQMRPDNSK*PPVYGA
302



misc.


CR



 


282
Enzyme,
FBXL11
808
AKIRGSYLTVTLQRPTK*
303



misc.






 


283
Enzyme,
FDPS
293
QILEENYGQK*DPEKVAR
304



misc.






 


284
Enzyme,
FDPS
297
QILEENYGQKDPEK*VAR
305



misc.






 


285
Enzyme,
FM03
209
VLVIGLGNSGCDIAAELSHV
306



misc.


AQK*V







TISSR



 


286
Enzyme,
Fmo5
259
NNYMEK*QMNQR
307



misc.






 


287
Unassigned
FUND2
123
SK*AEEVVSFVKKNVLVTGG
308






FFGG







FLLGMAS



 


288
Apoptosis
G6PI
226
TFTTQETITNAETAK*EWFLE
309






AAKD







PSAVAK



 


289
Mitochondria!
GAPDH;
212; 213;
GAAQNIIPASTGAAK*AVGK
310



protein
Gm10291;
195; 219;






Gm13882;
231






EG622339;







L00638833





 


290
Mitochondria!
GAPDH;
256; 331;
LEKPAKYDDIK*K
311



protein
LOC676923;
239; 259;






Gm13882;
275; 476






LOCI00043839;







L00638833;







L00675602





 


291
Enzyme,
GDA
133
TLK*NGTTTACYFGTIHTDSS
312



misc.


LILAEI







TDKFGQR



 


292
G protein or
G-
33
VSK*ASADLMSYCEEHAR
313



regulator
gamma(12)





 


293
Enzyme,
GLUL
95
KDPNK*LVLCEVFK
314



misc.






 


294
Enzyme,
GNMT
96
YALK*ER
315



misc.






 


295
Enzyme,
GSTA2;
141; 141;
VLK*SHGQDYLVGNR
316



misc.
GSTA5;
141






GSTA3





 


296
Enzyme,
GSTA3
64
SDGSLM#FQQVPMVEIDGM#
317



misc.


K*LV







QTK



 


297
Enzyme,
GSTA3
64
SDGSLMFQQVPM#VEIDGM#
318



misc.


K*LV







QTK



 


298
Enzyme,
GSTA3
64
SDGSLMFQQVPM#VEIDGMK*LVQ
319



misc.


TK



 


299
Enzyme,
GSTM1
198
ISAYMK*SSR
320



misc.






 


300
Enzyme,
GSTM1;
51; 51; 52
FK*LGLDFPNLPYLIDGSHK
321



misc.
GSTM5;







GSTM4





 


301
Enzyme,
GSTM1;
68; 68; 69
LGLDFPNLPYLIDGSHK*IT
322



misc.
GSTM5;

QSNAIL R





GSTM4





 


302
Enzyme,
GSTP1
127
ALPGHLK*PFETLLSQNQGG K
323



misc.






 


303
Unassigned
Gstt3
218, 229
AK*DM#PPLMDPALK*
324


 


304
Enzyme,
Gulo
332
AMLEAHPK*VVAHYPVEVR
325



misc.






 


305
Enzyme,
Gulo
332
AM#LEAHPK*VVAHYPVEV R
326



misc.






 


306
Chromatin,
H1F0
59
SHYK*VGENADSQIK
327



DNA-







binding,







DNA repair







or DNA







replication







protein






 


307
Unassigned
H2AE
126
VTIAQGGVLPNIQAVLLPKK
328






TESHH K*PK



 


308
Chromatin,
H2AX
127
KSSATVGPK*APAVGK
329



DNA-binding,







DNA repair or







DNA







replication







protein






 


309
Chromatin,
H2B;
5; 6; 6
PEPAK*SAPAPK
330



DNA-
H2B2E;






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*IGGHGAEYGAEAL
332



channel,


ER




transporter







or cell







surface







protein






 


312
Receptor,
HBA1
41
M#FASFPTTK*TYFPHFDVSH
333



channel,


GSAQ VK




transporter







or cell







surface







protein






 


313
Receptor,
HBA1
41
MFASFPTTK*TYFPHFDVSH
334



channel,


GSAQ VK




transporter







or cell







surface







protein






 


314
Receptor,
HBA1
57
TYFPHFDVSHGSAQVK*GHG
335



channel,







transporter







or cell






 


315
Receptor,
HBA1
91
VADALASAAGHLDDLPGA
336



channel,


LSALSD LHAHK*LR




transporter







or cell







surface







protein






 


316
Receptor,
HBA1
91
KVADALASAAGHLDDLPG
337



channel,


ALSALS DLHAHK*LR




transporter







or cell







surface







protein






 


317
Receptor,
HBB
17
SAVSCLWAK*VNPDEVGGE
338



channel,


ALGR




transporter







or cell







surface







protein






 


318
Receptor,
HBB
59
YFDSFGDLSSASAIMGNPK*
339



channel,







transporter







or cell






 


319
Receptor,
HBB
82
NLDNLK*GTFASLSELHCDK
340



channel,


LHVDP ENFR




transporter







or cell







surface






 


320
Receptor,
HBD
17
AAVSCLWGK*VNSDEVGGE
341



channel,







transporter







or cell






 


321
Receptor,
HBD
59
YFDSFGDLSSASAIM#GNAK*
342



channel,







transporter







or cell






 


322
Receptor,
HBD
82
VITAFNDGLNHLDSLK*GTF
343



channel,


ASLSEL HCDKLHVDPENFR




transporter







or cell






 


323
Enzyme,
HGD
71
ILPSVSHK*PFESIDQGHVTH
344



misc.


NWDE VGPDPNQLR



 


324
Enzyme,
HGD
252
FQGK*LFACK
345



misc.






 


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,
HPD
62
EVVSHVIK*QGK
349



misc.






 


329
Enzyme,
HPD
126
IVREPWVEQDK*FGK
350



misc.






 


330
Enzyme,
HPD
129
IVREPWVEQDKFGK*VK
351



misc.






 


331
Enzyme,
HPD
131
VK*FAVLQTYGDTTHTLVEK
352



misc.






 


332
Enzyme,
HPD
131
IVREPWVEQDKFGKVK*
353



misc.






 


333
Enzyme,
HPD
236
SIVVTNYEESIK*MPINEPAPG R
354



misc.






 


334
Enzyme,
HPD
247
K*KSQIQEYVDYNGGAGVQ
355



misc.


HIALK



 


335
Enzyme,
HPD
248
KK*SQIQEYVDYNGGAGVQ
356



misc.


HIALK



 


336
Enzyme,
HPD
269
SQIQEYVDYNGGAGVQHIAL
357



misc.


K*TED IITAIR



 


337
Enzyme,
HPD
296
ERGTEFLAAPSSYYK*LLR
358



misc.






 


338
Enzyme,
HPD
368
HNHQGFGAGNFNSLFK*AF
359



misc.


EEEQA LR



 


339
Enzyme,
HRSP12
66
NLGEILK*AAGCDFNNVVK
360



misc.






 


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*NQTAE
366






KEEFEH QQK



 


346
Chaperone
HSC70
583
ILDKCNEIISWLDK*NQTAE
367






KEEFEH 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
IPPK
42, 43, 64
K*K*TSEEILQHLQNIVDFGKNVMK*
372



(non-







protein)






 


352
G protein
IQGAP2
1024
AWVNQLETQTGEASK*LPY
373



or


DVTTE QALTYPEVK




regulator






 


353
G protein
IQGAP2
1354
TPEEGK*QSQAVIEDAR
374



or







regulator






 


354
RNA
IREB1
79
NIEVPFK*PAR
375



processing






 


355
Ubiquitin
ITCH
192
VSTNGSEDPEVAASGENK*R
376



conjugating







system






 


356
Ubiquitin
ITCH
407
FIYGNQDLFATSQNKEFDPL
377



conjugating


GPLPP GWEK*R




system






 


357
Unassigned
ITM2B
13
VTFNSALAQK*EAK
378


 


358
Unassigned
JOSD1
180
GK*NCELLLVVPEEVEAHQS
379






WR



 


359
Enzyme,
KHK
159
IEEHNAK*QPLPQK
380



misc.






 


360
Unknown
KIAA1033
1089
AVAK*QQNVOSTSQDEK
381



function






 


361
Cytoskeletal
lamin A/C
270
TYSAK*LDNAR
382



protein






 


362
Cytoskeletal
Lamin B1
124, 134
K*ESDLSGAQIK*LR
383



protein






 


363
Vesicle
LAPTM4A
224
IPEK*EPPPPYLPA
384



protein






 


364
Calcium-
LETM1
715
VIDLVNKEDVQISTTQVAEI
385



binding


VATLEK *EEK




protein






 


365
Receptor,
LISCH
538
LLEEALK*K
386



channel,







transporter







or cell







surface







protein






 


366
Unassigned
LOC1000444
63, 73;
MQANNAK*AVSARTEAIK*A
387




94; Gm12508
176, 186
LVK



 


367
Unassigned
LOCI000483
247
SYLLEK*IKEHEESLDVTNPR
388




23





 


368
Unassigned
LOCI000483
343
K*HMPYTNAMVHEVQR
389




23





 


369
Unassigned
LOC1000483
375
YVDLGPTSLVHEVTCDTK*F R
390




23





 


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*GEATAERPGEAAVAS
393



protein


SPSK



 


373
Enzyme,
MAT1A
54
QDPNAK*VACETVCK
394



misc.






 


374
Enzyme,
MAT1A
89
DTIK*HIGYDDSAK
395



misc.






 


375
Enzyme,
MAT1A
98
HIGYDDSAK*GFDFK
396



misc.






 


376
Enzyme,
MAT1A
352
ELLEVVNK*NFDLRPGVIVR
397



misc.






 


377
Enzyme,
MAT1A
368
DLDLK*KPIYQK
398



misc.






 


378
Enzyme,
MAT1A
374
KPIYQK*TACYGHFGR
399



misc.






 


379
Enzyme,
MAT1A
374
DLDLKKPIYQK*TACYGHFG R
400



misc.






 


380
Unassigned
MBD2
193
SDVYYFSPSGKKFRSK*
401


 


381
Unassigned
MCT1
467
EGKEDEASTDVDEK*PKETM
402


 


382
Unassigned
MCT1
469
EGKEDEASTDVDEKPK*ETM
403


 


383
Enzyme,
Mettl7b
241
WLPVGPHIM#GK*AVK
404



misc.






 


384
Enzyme,
Mettl7b
241
WLPVGPHIMGK*AVK
405



misc.






 


385
Enzyme,
MGST1
59
VFANPEDCAGFGKGENAK* K
406



misc.






 


386
Enzyme,
MGST1
60
VFANPEDCAGFGKGENAKK*
407



misc.






 


387
Transcriptional
MORF4L1
117
ELQK*ANQEQYAEGK
408



regulator






 


388
Mitochondrial
MOSC1
313
LCDPSEQALYGK*LPIFGQY
409



protein


FALEN PGTIR



 


389
Adaptor/
MPP5
553
DYHFVSRQAFEADIAAGKFI
410



scaffold


EHGEF EK*NLYGTSIDSVR



 


390
Receptor,
MT2A
20
MDPNCSCASDGSCSCAGAC
411



channel,


K*CK




transporter







or cell







surface







protein






 


391
Mitochondrial
MTX1
41
IHK*TSNPWQSPSGTLPALR
412



protein






 


392
Ubiquitin
NEDD8
54
QMNDEK*TAADYK
413



conjugating







system






 


393
Enzyme,
NGLY1
130
KVQFSQHPAAAK*LPLEQSE
414



misc.


DPAG LIR



 


394
Enzyme,
NGLY1
130
VQFSQHPAAAK*LPLEQSE
415



misc.


DPAGLI R



 


395
Enzyme,
NKEF-A
109
QGGLGPMNIPLISDPK*R
416



misc.






 


396
Kinase
NME2
56
QHYIDLK*DRPFFPGLVK
417



(non-







protein)






 


397
Adaptor/
NOSTRIN
417
AESK*APAGGQNNPSSSPSG
418



scaffold


STVS QASK



 


398
Enzyme,
NQ02
23
SFNGSLK*K
419



misc.






 


399
Vesicle
NSFL1C
127
GAK*EHGAVAVER
420



protein






 


400
Receptor,
NUP214
686
STQTAPSSAPSTGQK*SPRV
421



channel,


NPPV




transporter


PKSGSSQAKALQPPVTEK




or cell







surface







protein






 


401
Enzyme,
p67phox
354
EPKELKLSVPM#PYM#LK*
422



misc.






 


402
RNA
PABP 1
284
KFEQMK*QDR
423



processing






 


403
Enzyme,
PAH
49
EEVGALAK*VLR
424



misc.






 


404
Enzyme,
PAH
95
SKPVLGSIIK*SLR
425



misc.






 


405
Enzyme,
PAH
149
TIQELDRFANQILSYGAELD
426



misc.


ADHPG FK*DPVYR



 


406
Enzyme,
PAPSS2
174
AGEIK*GFTGIDSDYEKPET
427



misc.


PECVL K



 


407
Kinase
PCK1
124
WMSEEDFEK*AFNAR
428



(non-







protein)






 


408
Kinase
PCK1
124
WM#SEEDFEK*AFNAR
429



(non-







protein)






 


409
Kinase
PCK1
471
SEATAAAEHK*GK
430



(non-







protein)






 


410
Cell cycle
PCM-1
1089
QQNQHPEK*PR
431



regulation






 


411
Adaptor/
PDZK1
118
EAALNDKK*PGPGMNGAVE
432



scaffold


PCAQP R



 


412
Phosphatase
PGAM1
105
AETAAK*HGEAQVK
433


 


413
Vesicle
PICALM
324
EK*QAALEEEQAR
434



protein






 


414
Kinase
PIP5KG
97
GAIQLGIGYTVGNLSSK*PER
435



(non-







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






 


418
Receptor,
PMP70
576
EGGWDSVQDWMDVLSGGE
439



channel,







transporter







or cell






 


419
Enzyme,
PP ID;
285; 263
LQPIALSCVLNIGACKLK*
440



misc.
LOCI000452







51





 


420
Cytoskeletal
profilin 1
69
SSFFVNGLTLGGQK*CSVIR
441



protein






 


421
Protease
PSMA2
69
SVHKVEPITK*HIGLVYSGM
442






#GPDY R



 


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*EKAPSIIFIDELDAI
448



regulator


GTK



 


428
Protease
PSMC6
48
SENDLK*ALQSVGQIVGEVL
449


 


429
Protease
PSMC6
197
AVASQLDCNFLK*VVSSSIVD
450


 


430
Protease
PSMC6
197
AVASQLDCNFLK*VVSSSIVD
451






KYIGE 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,
PYGL
169
YEYGIFNQK*IR
455


 


435
Enzyme,
PYGL
803
AWNTM#VLK*NIAASGK
456


 


436
Enzyme,
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




LOCI000444
247; 148






94;







Gm1250





 


455
Unassigned
RPL29;
151; 151;
AQASAPAQAPKGAQAPK*
476




LOCI000444
264; 165






94;







Gm1250





 


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
YVASYLLAALGGNSSPSAKD
483


 


463
Enzyme,
RPN1
539
LK*TEGSDLCDRVSEMQK
484



misc.






 


464
Translation
RPS10
138
SAVPPGADK*K
485


 


465
Translation
RPS10
138
RSAVPPGADK*K
486


 


466
Translation
RPS10
138
SAVPPGADK*KAEAGAGSA
487






TEFQF R



 


467
Translation
RPS10
138, 139
SAVPPGADK*K*AEAGAGS
488






TEFQF R



 


468
Translation
RPS10
139
RSAVPPGADKK*
489


 


469
Translation
RPS10
139
SAVPPGADKK*AEAGAGSA
490






TEFQF R



 


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;
AEDK*EWIPVTK
494




Gm8841;
54






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*DEILPT
498






TPISEQ K



 


478
Translation
RPS3
214
IGPKKPLPDHVSIVEPK*DEI
499






LPTTPI SEQK



 


479
Translation
RPS3
230
GGK*PEPPAMPQPVPTA
500


 


480
Translation
RPS3a
45
NIGK*TLVTR
501


 


481
Translation
RPS7
10
IVK*PNGEKPDEFESGISQA
502






LLELE M#NSDLK



 


482
Translation
RPS7
15
IVKPNGEK*PDEFESGISQA
503






LLELE M#NSDLK



 


483
Translation
RPS7
15
IVKPNGEK*PDEFESGISQA
504






LLELE MNSDLK



 


484
Translation
RRBP1
145
K*VAKVEPAVSSIVNSIQVLA
505






SK



 


485
Translation
RRBP1
166
VEPAVSSIVNSIQVLASK*SA
506






ILEATP K



 


486
Translation
RRBP1
219, 249,
KGEGAQNQGK*KGEGAQNQ
507





289
AK



 


487
Translation
RRBP1
229, 299,
KGEGAQNQAK*KGEGAQNQ
508





359, 500
AK



 


488
Translation
RRBP1
259, 339,
KGEGAQNQAK*KGEGGQNQ
509





480




 


489
Translation
RRBP1
269
KGEGGQNQAK*KGEGAQNQ
510


 


490
Translation
RRBP1
279, 601,
KGEGAQNQGK*KGEGAQNQ
511





611, 621,







631, 641,







651, 681,




 


491
Translation
RRBP1
369, 510
KGEGAQNQAK*KGEGVQNQ
512


 


492
Translation
RRBP1
440, 581
IEGAQNQGK*KPEGTSNQGK
513


 


493
Translation
RRBP1
440, 581
KIEGAQNQGK*KPEGTSNQG
514


 


494
Translation
RRBP1
671
KGEGPQNQAK*KGEGAQNQ
515


 


495
Translation
RRBP1
752
TDTVANQGTK*QEGVSNQV
516


 


496
Translation
RRBP1
752
KTDTVANQGTK*QEGVSNQ
517


 


497
Translation
RRBP1
823
ASM#VQSQEAPK*QDAPAK
518


 


498
Translation
RRBP1
823
ASMVQSQEAPK*QDAPAK
519


 


499
Enzyme,
SAHH
46
EMYSASKPLK*GAR
520



misc.






 


500
Enzyme,
SAHH
166
GISEETTTGVHNLYK*M#MS
521



misc.


NGILK



 


501
Enzyme,
SAHH
166
GISEETTTGVHNLYK*MMSN
522



misc.


GILK



 


502
Enzyme,
SAHH
166
GISEETTTGVHNLYK*MM#S
523



misc.


NGILK



 


503
Enzyme,
SAHH
166
GISEETTTGVHNLYK*M#MS
524



misc.


NGILK VPAINVNDSVTK



 


504
Enzyme,
SAHH
166
GISEETTTGVHNLYK*MMS
525



misc.


NGILKV PAINVNDSVTK



 


505
Enzyme,
SAHH
166
GISEETTTGVHNLYK*M#M
526



misc.


#SNGIL KVPAINVNDSVTK



 


506
Enzyme,
SAHH
174
GISEETTTGVHNLYKM#MSN
527



misc.


GILK*



 


507
Enzyme,
SAHH
174
GISEETTTGVHNLYKMM#S
528



misc.


NGILK* VPAINVNDSVTK



 


508
Enzyme,
SAHH
186
VPAINVNDSVTK*SK
529



misc.






 


509
Enzyme,
SAHH
188
SK*FDNLYGCR
530



misc.






 


510
Adaptor/
SAKS1
105
MLELVAQK*QR
531



scaffold






 


511
Lipid
SEC14L2
11
VGDLSPK*QEEALAK
532



binding







protein






 


512
Lipid
SEC14L2
275
DQVK*QQYEHTVQVSR
533



binding







protein






 


513
Vesicle
SEC31L1
791
AQGK*PVSGQESSQSPYER
534



protein






 


514
Unassigned
SELENBP1;
342; 342
QYDISNPQK*PR
535




SELENBP2





 


515
Protein
SgK307
1148, 1153
NTSLTDIQDLSSITYDQDGYF
536



kinase,


K*ETS YK*TPKLK




Ser/Thr







(non-







receptor)






 


516
Chaperone
SGTA
161
AIGIDPGYSK*AYGR
537


 


517
Unassigned
SLC22A1
319
KVPPADLK*MMCLEEDASER
538


 


518
Receptor,
SLC26A1
32
ROPPVSQGLLETLK*AR
539



channel,







transporter







or cell







surface







protein






 


519
Endoplasmic
SLC27A5
163
LK*DAVIONTR
540



reticulum or







golgi






 


520
Endoplasmic
SLC27A5
599
VGMAAVK*LAPGK
541



reticulum or







golgi






 


521
Endoplasmic
SLC27A5
667
EGFDVGIIADPLYILDNK*AQ
542



reticulum or


TFR




golgi






 


522
Unassigned
S1c38a3
45
TEDTQHCGEGK*GFLQK
543


 


523
Unassigned
S1c38a3
50
GFLQK*SPSKEPHFTDFEGK
544


 


524
Unassigned
S1c38a3
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
KFTDEGNPEPVNNNGYSCV
548



channel,


PSDE 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*TGNA
551






GSR



 


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,
SULT1A1
93
IPFLEFSCPGVPPGLETLK*ET
555



misc.


PAPR



 


535
Enzyme,
SULT2A1
90
SPWIETDIGYSALINK*EGPR
556



misc.






 


536
Unassigned
SYNC1
37
M#ASPEPLRGGDGARASRE
557






PHTE ASFPLQESESPKEAK*



 


537
Adaptor/
SYNE2
5243
QSSLTM#DGGDVPLLEDMA
558



scaffold


SGIVE LFQK*K



 


538
Enzyme,
TALD01
258
ALAGCDFLTISPK*LLGELLK
559



misc.






 


539
Enzyme,
TALD01
265
LLGELLK*DNSK
560



misc.






 


540
Enzyme
TALD01
277
LAPALSVK*AAQTSDSEKIHL
561



misc.


DEK



 


541
Protein
Titin
855
ELSATSSTQK*ITK
562



kinase,







Ser/Thr







(non-







receptor)






 


542
Enzyme,
TKT
260
GITGIEDKEAWHGK*PLPK
563


 


543
Enzyme,
TKT
281
NMAEQIIQEIYSQVQSK*K
564


 


544
Unassigned
TMEM59
315
SQTEEHEEAGPLPTK*VNLAH
565


 


545
Vesicle
TOLLIP
143
lAWTHITIPESLK*QGQVEDE
566



protein


WYSL SGR



 


546
Unassigned
TRPM8
283, 298
NQLEK*YISERTSQDSNYGG
567






K*IPIV CFAQGGGRETLK



 


547
Cell cycle
TSGA2
35
NEVGERHGHGK*AR
568



regulation






 


548
Cytoskeletal
TUBB2C;
216; 216;
TLK*LTTPTYGDLNHLVSAT
569



protein
TUBB;
216; 216;
MSGVT TCLR





TUBB2A;
216






TUBB2B;







TUBB4





 


549
Enzyme,
TXNL1
180
LYSMK*FQGPDNGQGPK
570



misc.






 


550
Ubiquitin
UBE1
604
KPLLESGTLGTK*GNVQVVI
571



conjugating


PFLTE SYSSSQDPPEK




system






 


551
Ubiquitin
UBE1
627
GNVQVVIPFLTESYSSSQDPP
572



conjugating


EK*S IPICTLK




system






 


552
Ubiquitin
UBE1
635
SIPICTLK*NFPNAIEHTLQWAR
573



conjugating







system






 


553
Ubiquitin
Ube1y1;
184; 185
GIK*LVVADTR
574



conjugating
UBE1






system






 


554
Ubiquitin
UBE2D3;
128; 128
IYK*TDRDKYNR
575



conjugating
UBE2D4






system






 


555
Chromatin,
UBE2N
82
IYHPNVDK*LGR
576



DNA-







binding,







DNA repair







or DNA







replication







protein






 


556
Chromatin,
UBE2N
92
ICLDILK*DKWSPALQIR
577



DNA-







binding, DNA







repair or







DNA







replication







protein






 


557
Chromatin,
UBE2N
94
ICLDILKDK*WSPALQIR
578



DNA-







binding, DNA







repair or







DNA







replication







protein






 


558
Ubiquitin
UBE2Q1
216, 232,
K*SEDDGIGKENLAILEK*IK*
579



conjugating

234





system






 


559
Ubiquitin
ubiquitin;
113; 113
VDENGK*ISR
580



conjugating
L0C388720






system






 


560
Ubiquitin
ubiquitin;
113; 113
YYKVDENGK*ISR
581



conjugating
L0C388720






system






 


561
Ubiquitin
ubiquitin;
152; 152
CCLTYCFNK*PEDK
582



conjugating
L0C388720






system






 


562
Ubiquitin
UBQLN1
53
EKEEFAVPENSSVQQFK*EEI
583



conjugating


SKR




system






 


563
Enzyme,
UGP2
183
VK*IYTFNQSR
584



misc.






 


564
Mitochondrial
uricase
118
AHVYVEEVPWK*R
585



protein






 


565
Mitochondrial
uricase
220
DIVLQK*FAGPYDKGEYSPSV
586



protein


QK



 


566
Protease
USP33
227
SRPGSVVPANLFQGIK*TVNP
587






TFR



 


567
Protease
USP5
357
YVDK*LEKIFQNAPTDPTQD
588






FSTQV AK



 


568
Protease
USP5
360
YVDKLEK*IFQNAPTDPTQD
589






FSTQV AK



 


569
Protease
USP5
360
KYVDKLEK*IFQNAPTDPTQ
590






DFSTQ VAK



 


570
Protease
USP5
575
FASFPDYLVIQIKK*
591


 


571
Cytoskeletal
utrophin
50
SGK*PPISDM#FSDLKDGR
592



protein






 


572
Enzyme,
VARS
951
HFCNK*LWNATK
593



misc.






 


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#LIR
600



function


VANNIS K*



 


580
Transcriptional
YB-1
168
NYQQNYQNSESGEK*NEGS
601



regulator


ESAP EGQAQQR



 


581
Transcriptional
ZNF318
1246,
EVK*EDDK*APGELEEQLSE
602



regulator

1250,
DGSAP EK*GEVKGNASLR






1268





% 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 I5G15 protein because it is encoded by the I5G15 gene (see Blomstrom et al., J Biol Chem 261 (19): 8811-8816, 1986). Following digestion of such I5G15-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.

## Claims

1. 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.

2. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain sequence comprising SEQ ID NO: 1.

3. The isolated antibody of claim 1, wherein the antibody comprises a light chain sequence comprising SEQ ID NO: 2.

4. 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 the variable region of the heavy chain set forth in SEQ ID NO: 1.

5. 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 the variable region of the light chain set forth in SEQ ID NO: 2.