Abstract
Ubiquitination plays a key role in protein degradation and signal transduction. Ubiquitin is a small protein modifier that is adducted to lysine residues by the combined function of E1, E2, and E3 enzymes and is removed by deubiquitinating enzymes. Characterization of ubiquitination sites is important for understanding the role of this modification in cellular processes and disease. However, until recently, large-scale characterization of endogenous ubiquitination sites has been hampered by the lack of efficient enrichment techniques. The introduction of antibodies that specifically recognize peptides with lysine residues that harbor a di-glycine remnant (K-ε-GG) following tryptic digestion has dramatically improved the ability to enrich and identify ubiquitination sites from cellular lysates. We used this enrichment technique to study the effects of proteasome inhibition by MG-132 and deubiquitinase inhibition by PR-619 on ubiquitination sites in human Jurkat cells by quantitative high performance mass spectrometry. Minimal fractionation of digested lysates prior to immunoaffinity enrichment increased the yield of K-ε-GG peptides three- to fourfold resulting in detection of up to ~3300 distinct K-GG peptides in SILAC triple encoded experiments starting from 5 mg of protein per label state. In total, we identify 5533 distinct K-ε-GG peptides of which 4907 were quantified in this study, demonstrating that the strategy presented is a practical approach to perturbational studies in cell systems. We found that proteasome inhibition by MG-132 and deubiquitinase inhibition by PR-619 induces significant changes to the ubiquitin landscape, but that not all ubiquitination sites regulated by MG-132 and PR-619 are likely substrates for the ubiquitin-proteasome system. Additionally, we find that the proteasome and deubiquitinase inhibitors studied induced only minor changes in protein expression levels regardless of the extent of regulation induced at the ubiquitin site level. We attribute this finding to the low stoichiometry of the majority ubiquitination sites identified in this study.
Highlights
In our large-scale experiment, we investigated the usefulness of prefractionating samples prior to K--GG enrichment by comparing the number of K--GG peptides identified with limited Strong Cation Exchange (SCX) fractionation relative to direct enrichment
Three of our SILAC triple encoded samples (Fig. 2A) were divided such that 50% of the sample was directly enriched for K--GG peptides whereas the other 50% was first separated into four SCX fractions (Fig. 2B)
The workflow we developed employing limited prefractionation of peptides by SCX is compatible with K--GG enrichment and affords a three- to fourfold increase in the number of identified K--GG peptides in a given SILAC triple-encoded experiment
Summary
Aside from functioning to remove ubiquitin from substrate molecules, DUBs can function to trim polyubiquitin chains or generate free ubiquitin from ubiquitin precursors [5]. Proteasomal DUBs facilitate the recycling of cellular ubiquitin molecules upon substrate degradation [5]. Proteomic methods have utilized affinity-tagged ubiquitin, ubiquitin specific antibodies, or reagents consisting of ubiquitin binding domains to enrich substiochiometrically modified proteins [5,6,7]. These methods have enabled the characterization of up to several hundred sites of ubiquitination, they are inherently hampered by the increase in complexity that results from the presence of a large number of nonubiquitinated peptides derived from unmodified regions of the enriched proteins after digestion [9]. Technical improvements involving minimal fractionation of protein digests prior to liquid chromatography-tandem MS (LC-MS/MS) allowed us to obtain this level of coverage and quantification in SILAC triple encoded samples using a few milligram of protein lysate from each label state
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