Analysing non-proteinaceous ubiquitination via NoPro-clipping

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, a lack of suitable methods and reagents prevents reliable detection of these species and hinders us from understanding their true cellular significance.

Here we present NoPro-clipping, a mass spectrometry (MS)-based method for the unbiased detection of non-proteinaceous ubiquitination (see Figure). 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 use molecular weight filtration to enrich GlyGly-modified small molecules, including non-proteins, and analyse them by ammonium acetate/acetonitrile-based microflow liquid chromatography (LC)-MS/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 (e.g., Maltose, Ribose, GlcNAc) and is compatible with human cell lines and tissue samples. It can be applied in both targeted and untargeted studies, facilitating the detection of known and novel ubiquitinated non-proteins.

To unravel the underlying mechanisms of various human and horse glycogen storage diseases (e.g., McArdle's disease, Lafora disease, equine polysaccharide storage myopathy), we established liver cell lines in which we can observe and monitor the formation of ubiquitinated glycogen bodies via microscopy. Following cell lysis, α-amylase digestion, and NoPro-clipping, we successfully detected multiple ubiquitinated maltose species (e.g., GG‑maltose, GG‑maltotriose, GG‑maltotetraose), demonstrating that NoPro-clipping is capable of detecting ubiquitinated glycogen, which, to our knowledge, has not been previously reported in vivo.