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Determining Site-Specific Glycan Profiles of Recombinant SARS-CoV-2 Spike Proteins from Multiple Sources



Meghan Burke Harris, Yi Liu, Concepcion Remoroza, Yuri Mirokhin, Sergey Sheetlin, Dmitrii V. Tchekhovskoi, Guanghui Wang, Xiaoyu (Sara) Yang, Stephen E. Stein


Glycopeptide Abundance Distribution Spectra (GADS) were recently introduced as a means of representing, storing and comparing glycan profiles of intact glycopeptides. Here, using that representation, an extensive analysis is made of multiple commercial sources of recombinant SARS-CoV-2 spike protein, each containing 22 N-linked glycan sites (sequons). Multiple proteases are used along with variable energy fragmentation, followed by ion trap confirmation. This enables a detailed examination of reproducibility of the method across multiple types of variability. These results show that GADS are consistent between replicates and laboratories for sufficiently abundant glycopeptides. Then derived GADS enable the examination and comparison of the glycan profiles between commercial sources of the spike protein. Throughout multiple distinct glycopeptide distributions, generated by multiple proteases, confirm these profiles. Comparisons of GADS derived from 11 sources of recombinant spike protein reveal that sources for which protein expression methods were the same produced near-identical glycan profiles, thereby demonstrating the ability of this method to measure GADS of sufficient reliability to distinguish different glycoform distributions between commercial vendors and potentially to reliably determine and compare differences in glycosylation for any glycoprotein under different conditions of production.
ACS Journal of Proteome Research


glycopeptides, site-specific glycosylation, mass spectrometry, SARS-CoV-2, spectral library searching


Burke Harris, M. , Liu, Y. , Remoroza, C. , Mirokhin, Y. , Sheetlin, S. , Tchekhovskoi, D. , Wang, G. , Yang, X. and Stein, S. (2023), Determining Site-Specific Glycan Profiles of Recombinant SARS-CoV-2 Spike Proteins from Multiple Sources, ACS Journal of Proteome Research, [online], (Accessed June 15, 2024)


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Created August 30, 2023, Updated November 6, 2023