Towards structure-focused glycoproteomics

Abstract

Facilitated by advances in the separation sciences, mass spectrometry and informatics, glycoproteomics, the analysis of intact glycopeptides at scale, has recently matured enabling new insights into the complex glycoproteome. While diverse quantitative glycoproteomics strategies capable of mapping monosaccharide compositions of N- and O-linked glycans to discrete sites of proteins within complex biological mixtures with considerable sensitivity, quantitative accuracy and coverage have become available, developments supporting the advancement of structure-focused glycoproteomics, a recognised frontier in the field, have emerged. Technologies capable of providing site-specific information of the glycan fine structures in a glycoproteome-wide context are indeed necessary to address many pending questions in glycobiology. In this review, we firstly survey the latest glycoproteomics studies published in 2018-2020, their approaches and their findings, and then summarise important technological innovations in structure-focused glycoproteomics. Our review illustrates that while the O-glycoproteome remains comparably under-explored despite the emergence of new O-glycan-selective mucinases and other innovative tools aiding O-glycoproteome profiling, quantitative glycoproteomics is increasingly used to profile the N-glycoproteome to tackle diverse biological questions. Excitingly, new strategies compatible with structure-focused glycoproteomics including novel chemoenzymatic labelling, enrichment, separation, and mass spectrometry-based detection methods are rapidly emerging revealing glycan fine structural details including bisecting GlcNAcylation, core and antenna fucosylation, and sialyl-linkage information with protein site resolution. Glycoproteomics has clearly become a mainstay within the glycosciences that continues to reach a broader community. It transpires that structure-focused glycoproteomics holds a considerable potential to aid our understanding of systems glycobiology and unlock secrets of the glycoproteome in the immediate future.

Keywords: LC-MS/MS; glycopeptide; glycoproteins; glycoproteomics; glycosylation; mass spectrometry.

Figure 1.

Glycoproteome coverage reported by recent glycoproteomics studies published in the period 2018–2020 arranged by the studied species and publication year. The studies included in this overview all performed large-scale glycoproteomics analysis of complex biological samples from unaltered or only mildly manipulated biological sources and reported a significant number of intact glycopeptides. Studies not matching these criteria and studies reanalysing existing glycoproteomics data were left out. We apologise for any omission of studies published in this period that we may have failed to identify in our survey of the literature. The coverage as measured by the reported unique (non-redundant) intact N-glycopeptides (yellow bars) and O-glycopeptides (blue bars) has been plotted for each study. Details of these 28 glycoproteomics studies including their full references are provided in Table 1.

Figure 2.. Technical innovations supporting the advancement of structure-focused glycoproteomics.

The advances and the structural information they provide have been separated into sample preparation including protein digestion, glycoepitope labelling and enrichment, online separation and LC–MS/MS detection of intact N-glycopeptides (yellow) and O-glycopeptides (blue). See key for symbol and linkage nomenclature [105].