Analysis of ubiquitinated 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. A method of isolating peptides comprising lysine residues having a ubiquitin remnant comprising:
(a) digesting a sample of proteins with a hydrolyzing agent to produce a mixture of cleavage peptides;
(b) contacting the mixture of cleavage peptides with an antibody or a specific binding fragment thereof that specifically binds a ubiquitin remnant, wherein the antibody or specific binding fragment thereof comprising a heavy chain CDR1 as set forth in SEQ ID NO:3, a heavy chain CDR2 as set forth in SEQ ID NO:4, a heavy chain CDR3 as set forth in SEQ ID NO:5, a light chain CDR1 as set forth in SEQ ID NO:6, a light chain CDR2 as set forth in SEQ ID NO: 7, and a light chain CDR3 as set forth in SEQ ID NO:8;
(c) eluting cleavage peptides that have specifically bound to the antibody or specific binding fragment thereof; and
(d) purifying the cleavage peptides, such that peptides comprising lysine residues having a ubiquitin remnant are detected.
2. The method of claim 1 , wherein the proteins are a suspension of cells, a subcellular fraction of a cell line or tissue, or a cellular lysate.
3. The method of claim 1 , wherein the hydrolyzing agent is a protease.
4. The method of claim 3 , wherein the protease is trypsin.
5. The method of claim 1 , wherein the antibody or specific binding fragment thereof is linked to a bead, polystyrene matrix, protein G agarose beads, agarose gel matrix, or nitrocellulose membrane.
6. The method of claim 1 , wherein the antibody or specific binding fragment thereof is linked to an agarose bead.
7. The method of claim 1 , wherein the cleavage peptides are purified by HPLC or reversed phase liquid chromatography.
8. The method of claim 1 , wherein after the cleavage peptides are purified they are subjected to liquid chromatography MS/MS analysis.