Ubiquitin is an essential eukaryotic protein that can be covalently attached to other molecules in complex combinatory ways. It is involved in the regulation of almost every cellular process and therefore plays a role in a large number of human diseases, including various cancers, infections or neurodegenerative and autoimmune diseases. Recent studies have described the ubiquitination of lipopolysaccharides (LPS), sugars and lipids, expanding the role of ubiquitin modifications beyond proteins. However, methodologies to investigate non-protein ubiquitination have so far been hampered by a lack of suitable tools and reagents.
In this study, we explore non-proteinaceous ubiquitination through the development and use of a new, unbiased, mass spectrometry-based workflow. A key component of our method 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 analyze them by ammonium acetate/acetonitrile-based liquid chromatography (LC) connected to a quadrupole-orbitrap mass spectrometer (MS). Following rigorous data filtration and normalization, we conduct statistical analyses to pinpoint differentially measured masses specific to clippase-treated samples, thereby identifying previously ubiquitinated small molecules. GlyGly-characteristic mass-shifts, MS2 fragmentation spectra and tandem-MS repository databases are then used to infer the identity of intriguing molecules. At this stage, our method robustly detects in-vitro catalysed ubiquitinated sugars (Ub-maltose, Ub-maltoheptaose, Ub-LacNAc) that were spiked into lysates from various human cell lines.
The development of this protocol offers unprecedented potential to uncover novel biological mechanisms and pathways. Many key questions remain unanswered, and our method is designed to address them, for example, which other non-proteinaceous molecules can be ubiquitinated? What triggers their ubiquitination? How widespread does this phenomenon occur within cells and what is its clinical relevance?