Mass spectral feature analysis of ubiquitylated peptides provides insights into probing the dark ubiquitylome.

Ubiquitylation is a structurally and functionally diverse post translational modification that involves the covalent attachment of the small protein ubiquitin to other protein substrates. Trypsin-based proteomics is the most common approach for globally identifying ubiquitylation sites. However, we estimate that such methods are unable to detect ~40% of ubiquitylation sites in the human proteome - i.e., the dark ubiquitylome - including many important for human health and disease. In this meta-analysis of three large ubiquitylomic datasets, we performed a series of bioinformatic analyses to assess experimental features that could aid in uniquely identifying site-specific ubiquitylation events. Spectral predictions from Prosit were compared to experimental spectra of tryptic ubiquitylated peptides, revealing previously uncharacterized fragmentation of the diGly scar. Analysis of the LysC-derived ubiquitylated peptides reveal systematic, multidimensional peptide fragmentation, including diagnostic b-ions from fragmentation of the LysC ubiquitin scar. Comprehensively, these findings provide diagnostic spectral signatures of modification events that could applied to new analysis methods for non-tryptic ubiquitylomics.

Protein complex heterogeneity and topology revealed by electron capture charge reduction and surface induced dissociation.

We illustrate the utility of native mass spectrometry (nMS) combined with a fast, tunable gas-phase charge reduction, electron capture charge reduction (ECCR), for the characterization of protein complex topology and glycoprotein heterogeneity. ECCR efficiently reduces the charge states of tetradecameric GroEL, illustrating Orbitrap m/z measurements to greater than 100,000 m/z. For pentameric C-reactive protein and tetradecameric GroEL, our novel device combining ECCR with surface induced dissociation (SID) reduces the charge states and yields more topologically informative fragmentation. This is the first demonstration that ECCR yields more native-like SID fragmentation. ECCR also significantly improved mass and glycan heterogeneity measurements of heavily glycosylated SARS-CoV-2 spike protein trimer and thyroglobulin dimer. Protein glycosylation is important for structural and functional properties and plays essential roles in many biological processes. The immense heterogeneity in glycosylation sites and glycan structure poses significant analytical challenges that hinder a mechanistic understanding of the biological role of glycosylation. Without ECCR, average mass determination of glycoprotein complexes is available only through charge detection mass spectrometry or mass photometry. With narrow m/z selection windows followed by ECCR, multiple glycoform m/z values are apparent, providing quick global glycoform profiling and providing a future path for glycan localization on individual intact glycoforms.