The small protein ubiquitin dictates the cellular fate of other proteins via covalent modification and subsequent degradation by proteasomal or lysosomal pathways. But ubiquitin signalling has consequences beyond degradation and is involved in almost every cellular process. Consequently, it plays a role in most human diseases, including various cancers, infections, or neurodegenerative and autoimmune diseases. Recent studies have described the ubiquitination of lipopolysaccharides (LPS), sugars, lipids and nucleic acids, expanding the role of ubiquitin modifications beyond proteins. However, many aspects of non-proteinaceous ubiquitination remain elusive, and a lack of suitable methods prevents reliable detection of these species, hindering us from understanding their true cellular significance.
Here we explore non-proteinaceous ubiquitination through the development and application of a new, unbiased workflow called NoPro-clipping. A key component of NoPro-clipping is the use of viral and bacterial proteases, referred to as ‘clippases’, which cleave ubiquitin at its C-terminus and leave characteristic GlyGly marks on ubiquitinated substrates. Subsequently, we enrich GlyGly-modified small molecules, including non-proteins, and analyse them by liquid chromatography (LC) tandem mass spectrometry (MS). We further increase the sensitivity of NoPro-clipping by enzymatically attaching a small peptide onto GlyGly-modified molecules. This transformative step renders non-proteinaceous metabolite samples peptidic, allowing the subsequent use of conventional proteomics workflows for further enrichment and nanoflow LC-MS/MS analyses. NoPro-clipping has been validated with a variety of in vitro ubiquitinated sugars and is compatible with human cell lines and tissue samples.
To unravel the underlying mechanisms of different human and horse glycogen storage diseases (e.g., McArdle's disease, Lafora disease, equine polysaccharide storage myopathy), we use NoPro-clipping on various liver cell line systems and mouse organs. In combination with α-amylase digestion, we successfully detect multiple ubiquitinated maltose species (e.g., Ub‑maltose, Ub‑maltotriose), demonstrating that NoPro-clipping is capable of detecting ubiquitinated glycogen in vivo.