Fig. 4: Mass spectrometry-based workflows to detect branched ubiquitin chains.

Three different approaches to detect branched chains are illustrated. In the classical bottom-up approach, branched chains involving neighboring lysines can be detected, but all other types of branched chains are invisible due to the cleavage of branched peptides at intervening lysines or arginines (left). Mutation of the single arginine located between K48 and K63 of ubiquitin (Arg54) allows the detection of branched K48/K63 chains using classical bottom-up methods (middle). In principle, other types of branched chains can be detected in this manner. In the middle-down approach, ubiquitin chains are digested with trypsin under native conditions or cleaved after Arg74 with a site-specific protease (right). This approach leaves all possible combinations of branch points intact. Specific chain configurations can then be identified by tandem mass spectrometry. LC–MS liquid chromatography-mass spectrometry, SRM selected reaction monitoring, PRM parallel reaction monitoring, LC–MS/MS liquid chromatography-tandem mass spectrometry.