Conventional ubiquitination occurs via the attachment of ubiquitin to lysine residues of substrate proteins, but several studies have convincingly shown ubiquitination of non-protein substrates such as lipopolysaccharide and lipids. These studies suggest that the substrate repertoire of ubiquitin extends beyond protein regulation. However, the scope and role of non-proteinaceous ubiquitination remain elusive, hindered by an absence of suitable methodologies for detecting these unconventional species.
Here I will present the NoPro-clipping method for the detection of non-proteinaceous ubiquitination. NoPro-clipping uses two complementary enzymes—a ubiquitin-clippase and a transpeptidase for labelling of ubiquitin-clipped substrates—enabling untargeted detection of non-proteinaceous ubiquitination via mass spectrometry. Using the NoPro-clipping method, ubiquitinated glycogen is detectable in any glycogen-containing tissue. The presence of endogenous Ub-glycogen raises fundamental questions about its role in metabolic regulation, glycophagy and glycogen storage diseases. Moreover, this approach opens new avenues to explore ubiquitination of other macromolecules and their potential roles in various cellular processes and disease.