GlycoFASP: A Universal Method to Prepare Complex Mixtures for O-Glycoproteomic Analysis

Glycoproteomic analyses remain a significant challenge due to the considerable heterogeneity in the composition, linkage, and localization of glycosylation. These issues are further complicated in the study of O -glycosylation, which primarily occurs on serine or threonine residues, given the lack of a consensus peptide motif and the proximity of O -glycosites. Mass spectrometry (MS)-based analysis is the premier technique for analyzing O -glycoproteins,but often equires prior separation or enrichment. Variability in the properties of O-glycosylation has thus far prevented the development of universal glycoconjugate enrichments. In response to this problem, we developed GlycoFASP, a one-pot preparation method for O -glycoproteins requiring only 1 mg of sample and limited protein purification. Our method isolates O -glycopeptides through selective proteolysis of glycoproteins using proteases with dual peptide–glycan cleavage motifs, called O -glycoproteases, on a molecular-weight cutoff (MWCO) filter. It can be processed in a single day and provides enrichment as broad as all O -glycoproteins in a sample or as narrow as peptides bearing specific O -glycan structures. GlycoFASP regularly achieves depletion of nearly all non- O -glycosylated proteins, with ≥70% of the protein signal originating from O -glycoproteins in analyses of human serum, seminal fluid, and cell growth media. Our method was compared to other common enrichments, such as glycan-binding proteins (GBP), strong anion exchange (SAX-ERLIC), and another digestion-based strategy in analyses of human serum. GlycoFASP identified over twice as many O -glycopeptides as all other methods. Given its ease of use, reagent accessibility, and low cost, we expect this method to greatly accelerate glycoscience research by enabling nonglycoproteomic experts to perform these analyses.