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 microheterogeneous post-translational
modification (PTM) on proteins and lipids. The dynamic nature of glycosylation
can be studied through metabolic incorporation of non-natural sugars
into glycan epitopes and their detection using bio-orthogonal probes.
However, this approach possesses a significant drawback due to nonspecific
background reactions and ambiguity of non-natural sugar metabolism.
Here, we report a probe-free strategy for their direct detection by
glycoproteomics and glycomics using mass spectrometry (MS). The method
dramatically simplifies the detection of non-natural functional group
bearing monosaccharides installed through promiscuous sialic acid, N-acetyl-d-galactosamine (GalNAc) and N-acetyl-d-glucosamine (GlcNAc) biosynthetic pathways. Multistage
enrichment of glycoproteins by cellular fractionation, subsequent
ZIC-HILIC (zwitterionic-hydrophilic interaction chromatography) based
glycopeptide enrichment, and a spectral enrichment algorithm for the
MS data processing enabled direct detection of non-natural 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 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.