Mass Spectral Feature\nAnalysis of Ubiquitylated Peptides\nProvides Insights into Probing the Dark Ubiquitylome

Ubiquitylation is a structurally and functionally diverse\npost-translational\nmodification that involves the covalent attachment of the small protein\nubiquitin to other protein substrates. Trypsin-based proteomics is\nthe most common approach for globally identifying ubiquitylation sites.\nHowever, we estimate that such methods are unable to detect ∼40%\nof ubiquitylation sites in the human proteome, <i>i.e.</i>, “the dark ubiquitylome”, including many important\nfor human health and disease. In this meta-analysis of three large\nubiquitylomic data sets, we performed a series of bioinformatic analyses\nto assess experimental features that could aid in uniquely identifying\nsite-specific ubiquitylation events. Spectral predictions from Prosit\nwere compared to experimental spectra of tryptic ubiquitylated peptides,\nrevealing previously uncharacterized fragmentation of the diGly scar.\nAnalysis of the LysC-derived ubiquitylated peptides reveals systematic,\nmultidimensional peptide fragmentation, including diagnostic b-ions\nfrom fragmentation of the LysC ubiquitin scar. Comprehensively, these\nfindings provide diagnostic spectral signatures of modification events\nthat could be applied to new analysis methods for nontryptic ubiquitylomics.