Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling.

Nature Biotechnology
Guoqiang XuSamie R Jaffrey

Abstract

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.

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Methods Mentioned

BETA
ubiquitination
374
acetylation
affinity purification
transfection
immunoprecipitation
pulled
protein assay
Assay

Software Mentioned

NACCESS
Phenyx
DSSP
PANTHER
Motif
Spectrum Mill
Spectrum Mill Mill
Database for Annotation , Visualization and Integrated Discove...
PENCE Proteome Analyst
Mascot

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