Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling

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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 α-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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Figure 1: Generation of monoclonal antibodies that selectively recognize diglycine-modified lysines.
Figure 2: Profiling immunopurified ubiquitin remnant–containing peptides to identify ubiquitinated proteins.
Figure 3: Bioinformatic analysis of ubiquitinated proteins and ubiquitin-modified lysines.
Figure 4: Colchicine differentially regulates the ubiquitination of two lysines in PCNA.

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Acknowledgements

We thank T. Neubert and G. Zhang (New York University) for useful suggestions, P. Zhou (Weill Cornell Medical College, WCMC) for the His6-ubiquitin plasmid, U. Hengst, A. Deglincerti, R. Almeida and B. Derakhshan for the assistance during initial cell culturing, S. Gross and Y. Ma (WCMC Mass Spectrometry Core Facility) for helpful discussion in MS/MS analysis, F. Campagne, L. Skrabanek, J. Sun (WCMC Institute for Computational Biomedicine) for instructions and assistance in bioinformatic analysis. The mass spectrometry work was performed at the WCMC Mass Spectrometry Core Facility using instrumentation supported by US National Institutes of Health (NIH) RR19355 and RR22615. This work was supported by grants from Weill Cornell, NIH (MH086128) (S.R.J.), and a pharmacology cancer training grant from the National Cancer Institute (T32CA062948) (G.X. and J.S.P.).

Author information

S.R.J. and G.X. conceived and designed the study. G.X. and J.S.P. conducted the experiments, and G.X. and S.R.J. analyzed the data. S.R.J. and G.X. wrote the manuscript.

Correspondence to Samie R Jaffrey.

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