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.

Benefits of Chemical Sugar Modifications Introduced by Click Chemistry for Glycoproteomic Analyses

“…10,11 That said, the field of O-glycoproteomics has seen an explosion of technical advances in the last decade. 8,[11][12][13][14][15][16] Generally, glycoproteins are enriched using lectins, antibodies, and/or solid-phase extraction (i.e. hydrophilic interaction chromatography).…”

Recent Advances in Mass Spectrometry-Based Glycomic and Glycoproteomic Studies of Pancreatic Diseases

“…Glycoform quantification in disease states is a burgeoning area of research. The field has also been moving towards the frontier of "structure-focused glycoproteomics" as discussed in another recent review to analyze functional changes as imparted by structural modifications (Chernykh et al, 2021).…”

The Hitchhiker's guide to glycoproteomics

“…Understanding the molecular basis of how glycans are involved in health and disease requires technologies to precisely determine both the glycan structures (glycomics), and characterise their location and structure at discrete sites on glycoproteins (glycoproteomics) expressed by a cell or in an entire organ, body fluid, tissue or organism of interest. While the literature harbours many excellent technical reviews covering specific aspects of glycomics (such as [24][25][26][27][28][29][30][31][32][33][34]) and glycoproteomics (examples include [35][36][37][38][39][40][41][42][43][44]) technologies and methodologies, there is a gap in the literature surveying the methods and practical issues of modern glycoproteomics relevant to beginners in the field. This mini-review intends to provide a concise introduction to the current strategies available to generate glycoproteomics data and to provide some guidance for designing tailored glycoproteomics experiments.…”

“…It would go beyond this mini-review to provide a detailed account on the many different aspects of MS-based glycopeptide separation and detection, hence here we are focussing on a high-level overview of the most important advantages and challenges of widely used techniques. For more details, we refer readers to recent literature on this topic [38][39][40]44,142].…”

“…Given that glycopeptides are usually considerably larger and thus occur in higher charge states than unglycosylated co-eluting peptides, IM-MS provides an opportunity to separate glycopeptides from unglycosylated peptides within an online experiment using field asymmetric ion mobility spectrometry (FAIMS) [157]. While these recent technology developments have not yet found widespread application in glycoproteomics [44], promising data have been published for the characterisation of isolated glycoproteins [158][159][160], where IM-MS has been reported to be able to distinguish sialic acid linkage isomers (α2-3 or α2-6) from otherwise isobaric glycopeptide precursors [161], and to enable characterisation of isomeric glycopeptides where different sites on the same peptide are glycosylated [162].…”