Detection and quantification of modified proteins

Abstract

The invention provides a method detecting and quantifying proteins by mass spectrophotometric analysis using peptide internal standards and provides a highly sensitive way of detecting protein modifications. In one aspect, the invention provides a method for determining a site of ubiquitination in a polypeptide and for evaluating ubiquitination targets in a population of polypeptides. In this way, a proteome ubiquitination map can be obtained which comprises information relating to the ubiquitination states of a plurality of cellular polypeptides. Maps can be obtained for a variety of different types of cells and cell states. For example, ubiquitination targets in normal and diseased cells can be evaluated. Preferably, the map is stored as data files in a database. Individual ubiquitinated polypeptides identified can be used to generate molecular probes diagnostic of a cell state and/or can serve as targets for agents that modulate one or more cellular processes.

1 - 31 . (canceled) 
     
     
         32 . A kit comprising an antibody that specifically recognizes a peptide product of a protease-digested ubiquitinated protein which comprises a ubiquitin remnant. 
     
     
         33 . The kit according to  claim 32 , wherein the peptide comprises a lysine residue at position 6, 11, 27, 29, 33, 48, and 63 of the ubiquitin polypeptide. 
     
     
         34 . A kit comprising an antibody which specifically recognizes a ubiquitin polypeptide ubiquitinated at one or more of the K 6 , K 11 , K 27 , K 29 , K 33 , K 48 , and K 63  sites. 
     
     
         35 - 44 . (canceled) 
     
     
         45 . The kit according to  claim 32 , wherein the antibody comprises a monoclonal antibody or a portion thereof. 
     
     
         46 . The kit according to  claim 32 , wherein the antibody comprises a polyclonal antibody or a portion thereof. 
     
     
         47 . The kit according to  claim 32 , wherein the antibody is associated with a solid phase. 
     
     
         48 . The kit according to  claim 47 , wherein the solid phase is at least one selected from the group consisting of: a bead, a microparticle, a sphere, a chip, and a support. 
     
     
         49 . The kit according to  claim 47 , wherein the antibody is directly linked to the solid phase. 
     
     
         50 . The kit according to  claim 47 , wherein the antibody is chemically conjugated to the solid phase. 
     
     
         51 . The kit according to  claim 47 , wherein a binding partner links the antibody to the solid phase. 
     
     
         52 . The kit according to  claim 34 , wherein the antibody comprises a monoclonal antibody or a portion thereof. 
     
     
         53 . The kit according to  claim 34 , wherein the antibody comprises a polyclonal antibody or a portion thereof. 
     
     
         54 . The kit according to  claim 34 , wherein the antibody is associated with a solid phase. 
     
     
         55 . The kit according to  claim 54 , wherein the solid phase is at least one selected from the group consisting of: a bead, a microparticle, a sphere, a chip, and a support. 
     
     
         56 . The kit according to  claim 54 , wherein the antibody is directly linked to the solid phase. 
     
     
         57 . The kit according to  claim 54 , wherein the antibody is chemically conjugated to the solid phase. 
     
     
         58 . The kit according to  claim 54 , wherein a binding partner links the antibody to the solid phase. 
     
     
         59 . A kit comprising an antibody that specifically recognizes a peptide product of a protease-digested ubiquitinated protein, wherein the peptide product comprises a ubiquitin remnant. 
     
     
         60 . An antibody that specifically recognizes a peptide product of a protease-digested ubiquitinated protein which comprises a ubiquitin remnant. 
     
     
         61 . The antibody according to  claim 60 , wherein the ubiquitin remnant is attached to the peptide product.