Ubiquitin-Derived Diglycine Remnants as Chemical Handles for Chemoenzymatic Proteomics

Proteolytic processing is typically used to reveal post-translational modifications for mass spectrometric analysis. Here, we show that proteolysis of ubiquitinated proteins additionally generates a chemically addressable diglycine remnant that can be enzymatically remodeled. We exploit the ability of sortase to recognize ubiquitin-derived diglycine motifs as nucleophiles and ligate functionalized LPXTG peptides directly onto ubiquitin-remnant peptides. This reaction enables installation of a biotin affinity handle on defined ubiquitinated substrates and peptides derived from mammalian cell lysates, including lysine- and N-terminal ubiquitination. The resulting branched conjugates generate characteristic fragment ions that facilitate their identification. We further develop a bifunctional sortase donor containing an internal trypsin cleavage site, enabling affinity capture followed by conversion of enriched ubiquitin sites into a compact TGG mass-spectrometric signature. Alternatively, reversible sortase chemistry can regenerate the original diglycine-containing peptide. These studies establish ubiquitin-derived proteolytic remnants as latent chemical handles that can be converted into modular affinity and analytical tags, providing an antibody-independent approach to interrogating protein ubiquitination.