Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling.
Protein ubiquitination is a post-translational modification (PTM) that regulates various aspects of protein function by different mechanisms. Characterization of ubiquitination has lagged behind that of smaller PTMs, such as phosphorylation, largely because of the difficulty of isolating and identifying peptides derived from the ubiquitinated portion of proteins. To address this issue, we generated a monoclonal antibody that enriches for peptides containing lysine residues modified by diglycine, an adduct left at sites of ubiquitination after trypsin digestion. We use mass spectrometry to identify 374 diglycine-modified lysines on 236 ubiquitinated proteins from HEK293 cells, including 80 proteins containing multiple sites of ubiquitination. Seventy-two percent of these proteins and 92% of the ubiquitination sites do not appear to have been reported previously. Ubiquitin remnant profiling of the multi-ubiquitinated proteins proliferating cell nuclear antigen (PCNA) and tubulin alpha-1A reveals differential regulation of ubiquitination at specific sites by microtubule inhibitors, demonstrating the effectiveness of our method to characterize the dynamics of lysine ubiquitination.
Xu
Guoqiang
G
Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA.
Paige
Jeremy S
JS
Jaffrey
Samie R
SR
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods; D057809:HEK293 Cells / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D014404:Tubulin / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D057149:Ubiquitinated Proteins / Q000737:chemistry* / Q000378:metabolism*; D054875:Ubiquitination
D018809:Proliferating Cell Nuclear Antigen; D014404:Tubulin; D025801:Ubiquitin; D057149:Ubiquitinated Proteins; D008239:Lysine