Identification, mapping and relative quantitation of SARS-CoV-2 Spike glycopeptides by Mass-Retention Time Fingerprinting

Rod Chalk,Nicola A Burgess-Brown,Tina Bohstedt,William E P Greenland,Alejandra Fernandez-Cid,Tiago Moreira,Rama Mccrorie,Jesse Coker,Charlotte Manning,Shubhashish M M Mukhopadhyay,Eleanor Williams

doi:10.1038/s42003-021-02455-w

Rod Chalk, Nicola A Burgess-Brown + Show 9 more

Open Access

https://doi.org/10.1038/s42003-021-02455-w

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Abstract

We describe an analytical method for the identification, mapping and relative quantitation of glycopeptides from SARS-CoV-2 Spike protein. The method may be executed using a LC-TOF mass spectrometer, requires no specialized knowledge of glycan analysis and exploits the differential resolving power of reverse phase HPLC. While this separation technique resolves peptides with high efficiency, glycans are resolved poorly, if at all. Consequently, glycopeptides consisting of the same peptide bearing different glycan structures will all possess very similar retention times and co-elute. Rather than a disadvantage, we show that shared retention time can be used to map multiple glycan species to the same peptide and location. In combination with MSMS and pseudo MS3, we have constructed a detailed mass-retention time database for Spike glycopeptides. This database allows any accurate mass LC-MS laboratory to reliably identify and quantify Spike glycopeptides from a single overnight elastase digest in less than 90 minutes.

Highlights

We describe an analytical method for the identification, mapping and relative quantitation of glycopeptides from SARS-CoV-2 Spike protein
Any method, which is reliant on LC–MS–MS of glycopeptides, may not necessarily detect specific glycans that fail to fragment under the conditions selected
We have developed a simple Mass-Retention Time Fingerprinting (MRTF) method for mapping and relative quantitation of Spike glycopeptides

Summary

Introduction

We describe an analytical method for the identification, mapping and relative quantitation of glycopeptides from SARS-CoV-2 Spike protein. The current SARS-CoV-2 pandemic has spurred urgent research, much of it devoted to preparing vaccines, therapeutic antibodies, or antibody tests based on Spike protein, the virus’s primary surface antigen[4] This 145-kDa protein forms a trimer[5] with each subunit bearing twenty-two potential N-linked glycosylation sites and two O-linked sites of which approximately seventeen are occupied[5]. The resulting LC–MS data contain accurate mass, retention time and relative abundance values for each glycopeptide component. This dataset needs only to be matched against the preexisting Spike protein glycopeptide database reported here. We describe this method as “analytical mode”, which is both conceptually simple to understand, and straightforward to implement in any accurate mass LC–MS laboratory.

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