Abstract
Surface-embedded glycoproteins, such as the spike protein trimers of coronaviruses MERS, SARS-CoV, and SARS-CoV-2, play a key role in viral function and are the target antigen for many vaccines. However, their significant glycan heterogeneity poses an analytical challenge. Here, we utilized individual ion mass spectrometry (I2MS), a multiplexed charge detection measurement with similarities to charge detection mass spectrometry (CDMS), in which a commercially available Orbitrap analyzer is used to directly produce mass profiles of these heterogeneous coronavirus spike protein trimers under native-like conditions. Analysis by I2MS shows that glycosylation contributes to the molecular mass of each protein trimer more significantly than expected by bottom-up techniques, highlighting the importance of obtaining complementary intact mass information when characterizing glycosylation of such heterogeneous proteins. Enzymatic dissection to remove sialic acid or N-linked glycans demonstrates that I2MS can be used to better understand the glycan profile from a native viewpoint. Deglycosylation of N-glycans followed by I2MS analysis indicates that the SARS-CoV-2 spike protein trimer contains glycans that are more difficult to remove than its MERS and SARS-CoV counterparts, and these differences are correlated with solvent accessibility. I2MS technology enables characterization of protein mass and intact glycan profile and is orthogonal to traditional mass analysis methods such as size exclusion chromatography-multiangle light scattering (SEC-MALS) and field flow fractionation-multiangle light scattering (FFF-MALS). An added advantage of I2MS is low sample use, requiring 100-fold less than other methodologies. This work highlights how I2MS technology can enable efficient development of vaccines and therapeutics for pharmaceutical development.
Original language | English (US) |
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Pages (from-to) | 62-73 |
Number of pages | 12 |
Journal | Journal of the American Society for Mass Spectrometry |
Volume | 35 |
Issue number | 1 |
DOIs | |
State | Published - Jan 3 2024 |
Funding
The authors are particularly grateful to Dr. Michael W. Senko of Thermo Fisher Scientific for his scientific guidance in conducting the IMS experiments and for helpful feedback about this work. The authors also recognize the Merck & Co., Inc., Rahway, NJ, USA colleagues who contributed to the COVID-19 pandemic response programs. The authors thank Scott Cosmi, formerly of Eurofins Lancaster Laboratories Professional Scientific Service, Lancaster, PA, for his work in purifying the SARS-CoV and MERS proteins. The authors also appreciate Dr. Dustin Klein, Dr. Elizabeth Pierson, and Dr. Laurence Whitty-Léveillé for helpful scientific discussions. J.O.K. gratefully acknowledges funding support from National Institute of General Medical Sciences, Grant P41GM108569. 2 The authors are particularly grateful to Dr. Michael W. Senko of Thermo Fisher Scientific for his scientific guidance in conducting the I2MS experiments and for helpful feedback about this work. The authors also recognize the Merck & Co., Inc., Rahway, NJ, USA colleagues who contributed to the COVID-19 pandemic response programs. The authors thank Scott Cosmi, formerly of Eurofins Lancaster Laboratories Professional Scientific Service, Lancaster, PA, for his work in purifying the SARS-CoV and MERS proteins. The authors also appreciate Dr. Dustin Klein, Dr. Elizabeth Pierson, and Dr. Laurence Whitty-Léveillé for helpful scientific discussions. J.O.K. gratefully acknowledges funding support from National Institute of General Medical Sciences, Grant P41GM108569.
ASJC Scopus subject areas
- Structural Biology
- Spectroscopy