Fluorescent glycan fingerprinting of SARS2 spike proteins

Zhengliang L Wu,James M Ertelt

doi:10.1038/s41598-021-98919-4

Abstract

Glycosylation is the most common post-translational modification and has myriad of biological functions. However, glycan analysis has always been a challenge. Here, we would like to present new techniques for glycan fingerprinting based on enzymatic fluorescent labeling and gel electrophoresis. The method is illustrated on SARS2 spike (S) glycoproteins. SARS2, a novel coronavirus and the causative agent of the COVID-19 pandemic, has had significant social and economic impacts since the end of 2019. To obtain the N-glycan fingerprint of an S protein, glycans released from the protein are first labeled through enzymatic incorporation of fluorophore-conjugated sialic acid or fucose, then separated by SDS-PAGE, and finally visualized with a fluorescent imager. To identify the labeled glycans of a fingerprint, glycan standards and glycan ladders are enzymatically generated and run alongside the samples as references. By comparing the mobility of a labeled glycan to that of a glycan standard, the identity of glycans maybe determined. O-glycans can also be fingerprinted. Due to the lack of an enzyme for broad O-glycan release, O-glycans on the S protein can be labeled with fluorescent sialic acid and digested with trypsin to obtain labeled glycan peptides that are then separated by gel electrophoresis. Glycan fingerprinting could serve as a quick method for globally assessing the glycosylation of a specific glycoprotein.

Highlights

Glycosylation is the most common post-translational modification and has myriad of biological functions
O-glycans were not reported by Watanabe et al and Zhou et al These discrepancies could be due to the de facto variations on the glycosylations of the various recombinant spike proteins and different mass spectrometry methods employed in those studies, and highlight the necessity for additional methods of glycan verification
We have established a novel method for N-glycan fingerprinting based on enzymatic fluorescent glycan labeling and electrophoresis

Summary

Introduction

Glycosylation is the most common post-translational modification and has myriad of biological functions. Glycan fingerprinting could serve as a quick method for globally assessing the glycosylation of a specific glycoprotein. Glycan analysis is critical for researchers to better understand the biological functions of glycoproteins. Additional methods for glycan study that can be conveniently performed in common laboratories maybe beneficial. Mass spectrometry analysis revealed that the spike protein is highly modified with complex, hybrid, oligomannose N-glycans and more complicated N- and O-glycans[15,20,21,22]; the glycan profiles reported varied greatly. O-glycans were not reported by Watanabe et al and Zhou et al These discrepancies could be due to the de facto variations on the glycosylations of the various recombinant spike proteins and different mass spectrometry methods employed in those studies, and highlight the necessity for additional methods of glycan verification

Methods

Results

Conclusion