Proteomic Analysis of Ubiquitin Chain Types in Murine Cells and Tissues

ABSTRACT: Mass spectrometry-based proteomics has become an integral approach for characterising ubiquitin chain-linkage compositions and architectures. 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 (BMDMs) 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 ubiquitin chain types (K29 and K33) in heart and muscle. Ubiquitin chain topology profiling using tandem ubiquitin binding entities, directed at K29/K33 ubiquitin chains (TRABID), and mass spectrometry analysis identified several mitochondrial proteins as putatively 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. Our results contribute to our understanding of in vivo ubiquitin chain-linkage composition in murine tissues.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Spleen, Heart, Brain, Lung, Muscle, Bone Marrow, Kidney, Macrophage

SUBMITTER: Matthias Trost  

LAB HEAD: Matthias Trost

PROVIDER: PXD017754 | Pride | 2020-09-28

REPOSITORIES: Pride