UbiSite Approach: Global Site-Specific Profiling of Canonical and Non-Canonical Ubiquitination in Cells and Tissues

Ubiquitination is a complex post-translational modification that regulates a wide range of cellular processes through the covalent attachment of ubiquitin to substrate proteins. While canonical ubiquitination occurs on lysine residues, non-canonical modifications on serine, threonine, and the protein N-terminus are increasingly recognized. This chapter describes the UbiSite approach, a refined strategy for site-specific mapping of global ubiquitination using mass spectrometry-based proteomics. The method relies on digestion of the proteome of interest with LysC endopeptidase. LysC digestion leaves a 13 amino acid remnant on the ubiquitinated site of modified peptides, and this remnant is recognized by the monoclonal UbiSite antibody allowing selective enrichment of ubiquitinated peptides. Tens of thousands modification sites are readily identified from cells or tissue extracts in a single UbiSite experiment with the workflow described here. The 13 amino acid remnant is unique to ubiquitin and the UbiSite antibody thereby discriminates ubiquitin from other ubiquitin-like modifiers and enables detection of both canonical and non-canonical ubiquitination. The UbiSite procedure can be automated and combined with label-free quantification as well. UbiSite offers a robust and versatile platform for comprehensive ubiquitinome profiling with enhanced specificity and depth, enabling new insights into the regulatory roles of ubiquitin across diverse biological contexts.