Ubiquitin-Derived Diglycine Remnants as Chemical Handles for Chemoenzymatic Proteomics
- 1University of Pennsylvania
Abstract
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.
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BibTeX
@misc{raniszewski2026ubiquitinderived,
title = {{Ubiquitin-Derived Diglycine Remnants as Chemical Handles for Chemoenzymatic Proteomics}},
author = {N. Raniszewski and K. Beckley and J. Hintzen and M. Noel and G. Burslem},
howpublished = {bioRxiv},
year = {2026},
month = jun,
doi = {10.64898/2026.06.29.735432},
url = {https://www.biorxiv.org/content/10.64898/2026.06.29.735432v2},
note = {CC BY-ND},
}
RIS
TY - UNPB
TI - Ubiquitin-Derived Diglycine Remnants as Chemical Handles for Chemoenzymatic Proteomics
AU - N. Raniszewski
AU - K. Beckley
AU - J. Hintzen
AU - M. Noel
AU - G. Burslem
PY - 2026
DA - 2026/06/29
PB - bioRxiv
DO - 10.64898/2026.06.29.735432
UR - https://www.biorxiv.org/content/10.64898/2026.06.29.735432v2
AB - 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.
ER -