Ubiquitination (the attachment of a small protein tag called ubiquitin to cellular molecules) has long been understood as a mechanism for regulating protein function and degradation. However, mounting evidence suggested ubiquitin might also modify non-protein biomolecules such as sugars, lipids, and nucleotides. The tools to detect and quantify these modifications in living cells did not exist, leaving a significant gap in our understanding of ubiquitin biology.

To address this, Jochem et al. developed NoPro-clipping, a novel mass spectrometry workflow combining bacterial Ub-clippase enzymes with sortase-mediated peptide labelling to convert ubiquitinated non-protein molecules into species amenable to sensitive nanoflow LC-MS/MS analysis. For both nanoflow LC-MS/MS acquisition and absolute quantification (AQUA) of ubiquitin linkages via targeted AQUA mass spectrometry, samples were separated using a Vanquish Neo coupled to an Orbitrap Eclipse, with ClipTag-labelled analytes resolved on an Aurora® Elite™ 15×75 C18 UHPLC column. Targeted parallel reaction monitoring (PRM) was used for quantitative detection of specific ubiquitin-modified metabolite species, while untargeted MS1 acquisition enabled exploratory discovery of novel substrates.

Leveraging NoPro-clipping, the researchers from the Komander lab demonstrated that glycogen (the cell’s primary glucose storage molecule) is ubiquitinated in healthy mouse tissues, with ubiquitinated glycogen rising sharply during fasting before declining, pointing to an active role for ubiquitin in glycogen breakdown and lysosomal trafficking. The team further identified ubiquitinated glycerol and spermine as bona fide endogenous modifications, confirmed by metabolic labelling and biosynthetic inhibition.

These findings reframe ubiquitination as a broad cellular modification system extending well beyond proteins, with potential implications for glycogen storage diseases and metabolic disorders.

Publication
Nature

Authors

Marco Jochem, Simon A. Cobbold, Craig A. Goodman, Catharina Kueng, Anthony Cerra, Laura F. Fielden, Man Lyang Kim, Philipp Schenk, Ria Agarwal, Xiangyi S. Wang, Simon R. Scutts, Michael Pandos, Lin Tang, Thomas Hermanns, Shane M. Devine, Martin Brzozowski, Yuri Shibata, Niall D. Geoghegan, Catriona A. McLean, Bernhard C. Lechtenberg, Kay Hofmann, Paul Gregorevic, & David Komander;

Title

Ubiquitination of glycogen and metabolites in cells and tissues