ANALYSIS OF UBIQUITNATED POLYPEPTIDES
The disclosure 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.
1 . An isolated binding partner that 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.
2 . An isolated antibody that 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.
3 . The antibody of claim 2 , wherein the antibody is a monoclonal antibody.
4 . The antibody of claim 2 , wherein the antibody is a polyclonal antibody.
5 . The antibody of claim 2 , wherein the antibody is a human antibody.
6 . The antibody of claim 2 , wherein the antibody is a chimeric antibody.
7 . The antibody of claim 2 , wherein the antibody has a dissociation constant (kD) of 10 −7 M or less for the ubiquitin remnant peptide.
8 . The antibody of claim 2 , wherein the antibody has a dissociation constant (kD) of 10 −9 M or less for the ubiquitin remnant peptide.
9 . The antibody of claim 2 , wherein the antibody has an off rate (koff) of 10 −3 /sec or less.
10 . The antibody of claim 2 , wherein the antibody has an off rate (koff) of 10 −4 /sec or less.
11 . The antibody of claim 2 , wherein the antibody has an off rate (koff) of 10 −5 /sec or less.
12 . The antibody of claim 2 , wherein the antibody specifically binds a ubiquitinated lysine residue in the ubiquitin remnant peptide regardless of the peptide sequence surrounding the ubiquitinated lysine residue.
13 . The antibody of claim 2 , wherein the antibody is a monoclonal antibody.
14 . The antibody of claim 2 , wherein the ubiquitin remnant is a diglycine modified epsilon amine of lysine.
15 . The antibody of claim 2 , wherein the ubiquitin remnant is a portion of a ubiquitinated polypeptide that remains attached to a digestion product of the ubiquitinated polypeptide that has been exposed to trypsin.
16 . An isolated antibody that specifically binds to an amino acid modified with a gly-gly addition resulting from cleavage of a ubitquitin modification.
17 . The antibody of claim 16 , wherein the antibody is context-independent.