DISCUSSION
Comparing native and heat denatured SARS-CoV-2 spike demonstrated the utility of including deuterated glycopeptides in HDX-MS analyses of glycoprotein conformational dynamics. We conclude that 1) inclusion of 106 glycopeptides and 9 N-glycosylation sequons increased spike sequence coverage from 76% to 84%, 2) glycopeptides become deuterated and provide useful regional and protein-level information, 3) observed glycan identities are consistent with publications from non-deuterating studies (34, 39, 46), and 4) labeling of the amide nitrogens on N-acetyl groups of glycans merits inclusion in estimates of glycopeptide maximum deuteration.
Three sub-domains of spike glycoprotein which are known to have key functions during host cell infection by SARS-CoV-2 virus were adjacent to N-glycosylation sequons with confident deuterated glycopeptide coverage, including the 630 loop (N603), the S2’ cleavage site that releases the fusion peptide (N801), and the fusion peptide itself (N801). By detecting up to 37% deuteration in the control state in these portions of spike we corroborate existing HDX-MS descriptions of spike dynamics (1, 14). However, by adding information based on deuterated glycopeptides (N1134) we detected “hinge” motion between spike’s globular head and stalk, in addition to improved deuterated peptide resolution in the 630 loop, S2’ cleavage site, and fusion peptide. Using heat treatment to significantly change spike structure confirmed the utility of deuterated glycopeptide data by inducing a broader change in dynamics than a traditional HDX-MS state comparison such as binding to ACE2 (4, 14) or a monoclonal antibody (11, 21) would have induced. Pairing glycopeptide detection on a Tribrid Orbitrap Eclipse instrument with appropriate data analysis reveals dynamics of key spike sub-domains in proximity to N-glycosylation sequons, which has not been previously included in HDX-MS analyses (1, 4, 5, 13).
The identities of glycan groups that we detected at each N-glycosylation sequon were consistent with previous reports (33, 34, 39, 46), specifically with the most abundant glycans at each sequon. However, the microheterogeneity, or variety of glycan structures observed at each N-glycosylation sequon, for deuterated spike glycopeptides in this report did not indicate as many different glycan structures at each sequon as previously reported by others (39, 46), but this is not surprising given the differences in LC and MS/MS conditions in typical HDX-MS versus bottom-up glycopeptidomics analyses. For example, HDX-MS LC is typically conducted at relatively high flow rates (40 μL/min or higher, (58)) with large-bore chromatography columns (1.0 or 2.1 mm) and steep gradients (10 to 20 minutes) to minimize deuterium back-exchange (59). All these LC conditions are non-ideal for sensitive glycopeptide detection, which performs best at nano-flow rates (< 1 μL/min) with nano-electrospray ion sources and columns, and long gradients (1 to 2 hours). Given these limitations of HDX-MS LC, there is likely to be considerable room for improvement in the ionization, detection, and analysis of deuterated glycopeptides.
Analysis of product ions from deuterated glycopeptides supported previous reports (49, 50) of deuteration of N-acetyl hexose subunits of glycans attached to glycopeptides during HDX-MS analyses. The possibility of deuteron scrambling during HCD (51, 52) as the source of deuterated N-acetyl product ions cannot be excluded by our MS/MS data acquisition strategy, and a more targeted approach with ETD (53) or ultraviolet photo dissociation (UVPD) (54) MS/MS would be required. Deuteration of N-acetyl hexoses is significant because calculation of “maximum peptide deuteration” is based on the number of amino acid backbone amides that could have been labeled (all except the N-terminal 1 or 2 residues and any proline residues (60, 61)), so the presence of each N-acetyl group in a glycan structure could add one potential labeling site per glycopeptide. We are not aware of any HDX-MS data processing software that includes options for the deuteration of glycans attached to glycopeptides. Additional complexity is added by microheterogeneity, because glycans that differ in the number of N-acetyl groups will have different numbers of potential labeling sites. We will continue to explore the effect of glycan subunit composition on the maximum peptide deuteration level for different types of glycopeptides.
Inclusion of deuterated glycopeptides in HDX-MS is a step forward in attempts to measure glycoprotein dynamics under conditions that are as native as possible. Direct detection of deuterated glycopeptides from glycoproteins such as viral surface antigens and cellular receptors avoids additional HDX-MS procedures, for example de-glycosylation with PNGase before or after deuteration using acid-tolerant isoforms Rc (62), or H (63, 64). De-glycosylation adds time and complexity to sample preparation and potentially introduces additional back-exchange and/or artifacts during HDX-MS analyses. In the case of SARS-CoV-2 spike, previous reports indicate that glycans participate in modulation of the RBD moving to “up” or “down” positions (65), as well as in ACE2 interaction (66, 67), indicating the importance of measuring spike glycopeptide dynamics in a native state using HDX-MS.
Author Contributions
D.W., T.K. and C.H. conceptualized the study. S.O. prepared spike protein and B.M. performed heat-treatment. C.H. and T.K. performed HDX-MS. C.H. wrote the manuscript and all authors contributed to its review and editing.
Notes The authors declare no competing financial interest. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Use of trade names and commercial sources in this presentation is for identification only and does not imply endorsement by the Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, the Public Health Service, or the U.S. Department of Health and Human Services.
ACKNOWLEDGMENT
We thank Dr. Bin Zhou for the plasmids expressing the spike constructs used in this work.
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