Using the Ubiquitin-modified Proteome to Monitor Protein Homeostasis Function

Abstract

The ubiquitin system is essential for the maintenance of proper protein homeostasis function across eukaryotic species. Although the general enzymatic architecture for adding and removing ubiquitin from substrates is well defined, methods for the comprehensive investigation of cellular ubiquitylation targets have just started to emerge. Recent advances in ubiquitin-modified peptide enrichment have greatly increased the number of identified endogenous ubiquitylation targets, as well as the number of sites of ubiquitin attachment within these substrates. Herein we evaluate current strategies using mass-spectrometry-based proteomics to characterize ubiquitin and ubiquitin-like modifications. Using existing data, we describe the characteristics of the ubiquitin-modified proteome and discuss strategies for the biological interpretation of existing and future ubiquitin-based proteomic studies. Molecular & Cellular

Identification of Candidate Substrates for the Golgi Tul1 E3 Ligase Using Quantitative diGly Proteomics in Yeast

“…Tryptic digestion efficiency was assessed via SDS-PAGE prior to processing. A mixture of tryptic peptide samples was acidified with trifluoroacetic acid (TFA) to a final concentration of 1% and subsequently desalted using a C 18 Sep-Pak SPE cartridge (Waters, Milford, MA). C 18 cartridges were conditioned with 3 ml of 80% acetonitrile (ACN) followed by 3 ml of 50% ACN (0.1% TFA), and finally 5 ml of 0.1% TFA.…”

“…In total, we identified 3116 distinct K--GG peptides in 1111 proteins (supplemental Table S2) and obtained a heavy-to-light (H/L) ratio in at least one experiment for 2896 ubiquitin-modified peptides (93% of the total). Multiple studies have identified ubiquitylated peptides in S. cerevisiae, with the recent application of quantitative diGly proteomics yielding thousands of ubiquitylated peptides per study (18,22,23). To evaluate our methodology, we compared our results to a published study that combined ubiquitylated protein enrichment followed by diGly proteomics without SILAC (22).…”

“…Recent technical advances enable enrichment of ubiquitylated peptides and greatly improve detection of this post-translational modification (18). Antibodies that recognize the diGly remnant on ubiquitin-conjugated lysine residues after trypsin digestion now allow the identification of thousands of ubiquitylated peptides via MS (19 -21).…”

Using the Ubiquitin-modified Proteome to Monitor Distinct and Spatially Restricted Protein Homeostasis Dysfunction

“…Proteomic approaches can interrogate the abundance of the endogenous ubiquitin-modified proteome and offer a more unbiased and comprehensive solution to the limitations of using single model substrates to report on UPS activity. Immunoaffinity isolation of the diGlycine (diGLY) ubiquitin remnant remaining after tryptic digestion of ubiquitylated proteins can be utilized to identify and quantify the ub-modified proteome (22,44). Even though this approach does not unambiguously identify ubiquitylated proteins, we will refer to the resulting data as the ub-modified proteome because of the observation that over 94% of all diGLY-modified proteins arise from ubiquitylation events (23).…”

“…The development of quantitative proteomic approaches to interrogate the ubiquitin (ub) 1 -modified proteome provides an opportunity to globally monitor protein homeostasis function without exogenous expression of reporter proteins (22,23). One advantage of the ub-proteomics approach is the ability to interrogate a wide-array of endogenous ubiquitylation events that either target proteins for degradation or regulate protein function without proteasomal targeting.…”