Conjugated ubiquitin and polyubiquitins play critical but incompletely understood roles in cell signaling and cell biology. For the mass spectrometrist these conjugates offer the challange/opportunity to extend top-down proteomic strategies to the analysis of branched proteins.. We have succeeded in trapping sufficient numbers of ubiquitin trimer ions (25.5 kDa), tetramer ions (34 kDa) and pentamer ions (42.5 kDa) using a tribrid mass spectrometer, and obtained sufficiently dense fragmentation using combinations of ETD and CID (EThcD) to provide MS/MS spectra that contain the required structural information, including sites of conjugation and topology. We have facilitated interpretation of these spectra with a novel use of existing software. Our standards have been custom-synthesized to provide an assortment of topologies. HPLC was carried out on a monolith column that provided moderate separation. Currently, an extended strategy is being tested to characterize ubiquitin chains conjugated to target proteins.

Progress in top-down analysis of branched proteins

253rd National Meeting (2017)

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