diGly

diGly, in proteomics, refers to the diglycine remnant that remains on a lysine residue of a substrate protein after trypsin digestion in ubiquitin-like modification workflows. Ubiquitination involves attaching ubiquitin to lysine side chains through an isopeptide bond. When samples are proteolytically digested with trypsin, most of the ubiquitin is removed, but the C-terminal glycine residues of ubiquitin leave behind a di-glycine tag on the modified lysine. This diGly tag serves as a molecular signature indicating that the lysine was previously ubiquitinated.

In practice, diGly is detected by targeted mass spectrometry workflows. The presence of the diGly remnant produces a characteristic mass shift, approximately 114 Da, on the modified lysine, enabling site-specific identification within tryptic peptides. Enrichment strategies using anti-diGly antibodies are commonly employed to isolate diGly-containing peptides from complex mixtures, increasing the sensitivity for ubiquitination site mapping.

diGly-based proteomics has become a foundational approach for characterizing the ubiquitylome, aiding investigations into protein stability, signaling, DNA repair, and stress responses. It allows researchers to quantify changes in ubiquitination across conditions and to map site-specific regulatory networks.

Limitations include the potential for misassignment due to other ubiquitin-like modifiers that can leave similar remnants, sample preparation biases, and the need for careful validation of identified sites. Ongoing refinements aim to improve specificity, coverage, and quantitative accuracy in diGly workflows.