Mass Spectrometric Method for the Unambiguous Profiling of Cellular Dynamic Glycosylation

Abstract

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 glycosylation can be studied through bio-orthogonal tagging of metabolically engineered nonnatural sugars into glycan epitopes. However, this approach possesses a significant drawback due to nonspecific background 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 dramatically simplifies the detection of non-natural functional group bearing monosaccharides installed through promiscuous sialic acid, GalNAc, and GlcNAc biosynthetic pathways. Multistage enrichment of glycoproteins by cellular fractionation, subsequent ZIC-HILIC based glycopeptide enrichment, and a spectral enrichment algorithm for the MS data processing enabled direct detection of nonnatural monosaccharides that are incorporated at low abundance on the N/O-glycopeptides along with their natural counterparts. 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 incorporation 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 engineering in glycosylation functional studies.

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

“…In MOE experiments, we and others have observed that chemically modified GalNAc analogs can be elaborated by downstream glycosylation. [27][28][29]36,37 As demonstrated in Figure 2E and Supporting Information Figure S3A, B, extension of the GalNAc analogs GalN6yne, GalNAz, and GalNAzMe could be observed in our MOE systems by virtue of defined HCD fragment ions. It is currently not known whether downstream glycosylation of these analogs proceeds with the same fidelity as for native GalNAc.…”

“…However, recent findings suggest that monosaccharide analogs are incorporated into various glycan sub-types without a notable chain-terminating effect. 28,29,36,37…”

Optimization of Metabolic Oligosaccharide Engineering with Ac4GalNAlk and Ac4GlcNAlk by an Engineered Pyrophosphorylase

“…Epimerization substantially decreases the glycan specificity while enhancing the labeling efficiency of certain MOE reagents and can be suppressed by careful choice of the chemical modification. 10 − 12 Once biosynthesized, the derivatives of UDP-GalNAc and UDP-GlcNAc can be used as substrates by cellular GTs, including the large polypeptide GalNAc transferase (GalNAc-T) family in the secretory pathway and a myriad of GlcNAc transferases in several cellular compartments.…”

Design and synthesis of metabolic chemical reporters for the visualization and identification of glycoproteins

“…This is useful for studying endogenous sugar metabolism. 110 However, it complicates most applications of MCRs. Fortunately, individual salvage pathways exhibit some amount of substrate selectivity.…”

“…This was exemplified in recent work from the Kohler lab. 110 Here, efforts to generate a probe-free technique for the identification of glycoproteins illuminated the vast metabolic cross talk that interconverts GalNaz, GlcNAz and ManNAz. Additionally, there is still no universal MS workflow that generates site, structure, and biological context in one mega experiment.…”