Targeted Proteomic Analysis Reveals Enrichment of Atypical Ubiquitin Chains in Contractile Tissues

Ubiquitylation is an elaborate post-translational modification involved in all biological processes. Its pleotropic effect is driven by the ability to form complex polyubiquitin chain architectures that can influence biological functions. In this study, we optimised sample preparation and chromatographic separation of Ubiquitin peptides for Absolute Quantification by Parallel Reaction Monitoring (Ub-AQUA-PRM). Using this refined Ub-AQUA-PRM assay, we were able to quantify all ubiquitin chain types in 10-minute LC-MS/MS runs. We used this method to determine the ubiquitin chain-linkage composition in murine bone marrow-derived macrophages and different mouse tissues. We could show tissue-specific differences in ubiquitin levels in murine tissues, with polyubiquitin chain types contributing a small proportion to the total pool of ubiquitin. Interestingly, we observed enrichment of atypical (K29 and K33) ubiquitin chains in heart and muscle. Ubiquitin profiling using tandem ubiquitin binding entities, directed at K29/K33 ubiquitin chains, and mass spectrometry analysis identified several mitochondrial proteins associated with these atypical ubiquitin chain types. Our approach enabled high-throughput screening of ubiquitin chain-linkage composition in different murine tissues and highlighted a possible role for atypical ubiquitylation in contractile tissues, likely associated with mitochondrial function. Our results contribute to our understanding of in vivo ubiquitin chain-linkage composition in murine tissues.