Mass spectrometric method for the unambiguous profiling of cellular dynamic glycosylation.

Various biological processes at the cellular level are regulated by glycosylation which is a highly micro-heterogeneous post-translational modification (PTM) on proteins and lipids. The dynamic nature of gly-cosylation can be studied through bio-orthogonal tagging of metabolically engineered non-natural sugars into glycan epitopes. However, this approach possesses a significant drawback due to non-specific back-ground reactions and ambiguity of non-natural sugar metabolism. Here we report a tag-free strategy for their direct detection by glycoproteomics and glycomics using mass spectrometry. The method dramati-cally simplifies the detection of non-natural functional group bearing monosaccharides installed through promiscuous sialic acid, GalNAc, and GlcNAc biosynthetic pathways. Multistage enrichment of glyco-proteins by cellular fractionation, subsequent ZIC-HILIC based glycopeptide enrichment, and a spectral enrichment algorithm for the MS data processing enabled direct detection of non-natural monosaccha-rides that are incorporated at low abundance on the N/O-glycopeptides along with their natural counter-parts. Our approach allowed the detection of both natural and non-natural sugar bearing glycopeptides, N and O- glycopeptides, differentiation of non-natural monosaccharide types on the glycans and also their incorporation efficiency through quantitation. Through this we could deduce some interconversion of monosaccharides during their processing through glycan salvage pathway and subsequent incorpora-tion into glycan chains. The study of glycosylation dynamics through this method can be conducted in high throughput as few sample processing steps are involved, enabling understanding of glycosylation dynamics under various external stimuli and thereby could bolster the use of metabolic glycan engineer-ing in glycosylation functional studies.