Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry Enables Characterization of Branched Ubiquitin Chains in CellulO

摘要

Ubiquitin (Ub) has a broad functional range that has been ascribed to the formation of an array of polymeric ubiquitin chains. Understanding the precise roles of ubiquitin chains, however, is difficult due to their complex chain topologies. Branched ubiquitin chains are particularly challenging, as multiple modifications on a single ubiquitin preclude the use of standard bottom-up proteomic approaches. Developing methods to overcome these challenges is crucial considering evidence suggesting branched chains regulate the stability of proteins. In this study, we employ Ubiquitin Chain Enrichment Middle-down Mass Spectrometry (UbiChEM-MS) to identify branched chains that cannot be detected using bottom-up proteomic methods. Specifically, we employ tandem ubiquitin binding entities (TUBEs) and the K29-selective Np14 Zinc Finger 1 (NZF1) domain from the deubiquitinase TRABID to enrich for chains from human cells. Minimal trypsinolysis followed by high resolution mass spectrometric analysis-reveals that Ub chain branching can indeed be detected using both Ub binding domains (UBDs) tested at endogenous levels. We find that similar to 1% of chains isolated with TUBEs contain Ub branch points, with this value rising to similar to 4% after proteasome inhibition. Electron transfer dissociation (ETD) analysis indicates the presence of K48 in these branched chains. The use of the NZF1 domain reveals that similar to 4% of the isolated chains contain branch points with no apparent dependence on proteasome inhibition. Our results demonstrate an effective strategy for detecting and characterizing the dynamics of branched conjugates under different cellular conditions.