Characterizing Ub Chain Architecture

Characterizing the architecture of chains is difficult, as there is a dearth of methods for measuring chain length and the extent of chain branching. The reason for the latter is that branching likely occurs with a lower frequency relative to linear chain formation and involves multiple modifications on a single Ub, which precludes the use of standard bottom-up proteomic methods. To address these limitations, we sought to develop an alternative approach—one called middle-down proteomics. The middle-down method combines limited proteolysis with intact protein mass spectrometry to identify multiple modifications on Ub. In our initial studies, we used middle-down MS to characterize Ub chains produced by bacterial E3 ligases. We then extended the middle-down approach to detect endogenous branched chains in human cell extracts. The strategy, which is called Ubiquitin Chain Enrichment Middle-down Mass Spectrometry (UbiChEM-MS), couples Ub binding domains (UBDs) with middle-down MS. Using this strategy, wefound that the cellular levels of Lys29/Lys48 branched chains are unaffected by proteasome inhibition, whereas Lys11/Lys48 branched chains are highly dynamic and accumulate at different stages of the cell cycle. UbiChEM-MS opens the door to understanding the abundance and dynamics of branched chains in any biological paradigm. To further advance the methodology, we are designing capture reagents that can specifically enrich branched chains from cell extracts. We are also using middle-down MS to explore the extent to which other modifications occur on Ub chains under different cellular events, e.g., phosphorylation, acetylation, and SUMOylation.