Figure 1.

Approaches to detect polyubiquitin chains. (A) The polyubiquitin chains include homotypic and heterotypic chains where heterotypic chains are further divided into mixed and branched chains. (B) A gel-based approach uses either chain-specific antibody with Western blotting or the UbiCRest deubiquitinase (DUB) library to digest specific chain types. Either approach can detect and provide information of specific chain types from target sample, however, it cannot distinguish branched chain from mixed chains. (C) Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry (UbiChEM-MS) and Ub-clipping methods use minimal trypsinolysis or viral Lb protease (Lb ^pro) to generate Ub₁₋₇₄ with various di-Gly modifications, respectively. The Ub₁₋₇₄ variants (denoted as a small u symbol) could be detected by intact mass (MS) to reveal direct evidence of branched linkages (group A), unbranched linkages (group B), and end-point ubiquitin (group C). The linkage types of branched polyubiquitin chain can be analyzed by LC-MS/MS coupled with trypsinolysis. The combination of two MS-based analyses could offer a better understanding on the topology of a branched polyubiquitin chain.