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.
REFERENCES
1. Braet SM, Buckley TSC, Venkatakrishnan V, Dam KA, Bjorkman PJ, Anand
GS. Timeline of changes in spike conformational dynamics in emergent
SARS-CoV-2 variants reveal progressive stabilization of trimer stalk
with altered NTD dynamics. Elife. 2023;12.
2. Mehra R, Kepp KP. Structure and Mutations of SARS-CoV-2 Spike
Protein: A Focused Overview. ACS Infect Dis. 2022;8(1):29-58.
3. Alabsi S, Dhole A, Hozayen S, Chapman SA. Angiotensin-Converting
Enzyme 2 Expression and Severity of SARS-CoV-2 Infection.
Microorganisms. 2023;11(3).
4. Raghuvamsi PV, Tulsian NK, Samsudin F, Qian X, Purushotorman K, Yue
G, et al. SARS-CoV-2 S protein:ACE2 interaction reveals novel allosteric
targets. Elife. 2021;10.
5. Narang D, James DA, Balmer MT, Wilson DJ. Protein Footprinting,
Conformational Dynamics, and Core Interface-Adjacent Neutralization
”Hotspots” in the SARS-CoV-2 Spike Protein Receptor Binding Domain/Human
ACE2 Interaction. J Am Soc Mass Spectrom. 2021;32(7):1593-600.
6. Federico M. How Do Anti-SARS-CoV-2 mRNA Vaccines Protect from Severe
Disease? Int J Mol Sci. 2022;23(18).
7. Corbett KS, Edwards DK, Leist SR, Abiona OM, Boyoglu-Barnum S,
Gillespie RA, et al. SARS-CoV-2 mRNA vaccine design enabled by prototype
pathogen preparedness. Nature. 2020;586(7830):567-71.
8. Jackson CB, Farzan M, Chen B, Choe H. Mechanisms of SARS-CoV-2 entry
into cells. Nat Rev Mol Cell Biol. 2022;23(1):3-20.
9. Xia S, Liu M, Wang C, Xu W, Lan Q, Feng S, et al. Inhibition of
SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent
pan-coronavirus fusion inhibitor targeting its spike protein that
harbors a high capacity to mediate membrane fusion. Cell Res.
2020;30(4):343-55.
10. Zhao MM, Zhu Y, Zhang L, Zhong G, Tai L, Liu S, et al. Novel
cleavage sites identified in SARS-CoV-2 spike protein reveal mechanism
for cathepsin L-facilitated viral infection and treatment strategies.
Cell Discov. 2022;8(1):53.
11. Silva RP, Huang Y, Nguyen AW, Hsieh CL, Olaluwoye OS, Kaoud TS, et
al. Identification of a conserved S2 epitope present on spike proteins
from all highly pathogenic coronaviruses. Elife. 2023;12.
12. Vinciauskaite V, Masson GR. Fundamentals of HDX-MS. Essays Biochem.
2023;67(2):301-14.
13. Calvaresi V, Wrobel AG, Toporowska J, Hammerschmid D, Doores KJ,
Bradshaw RT, et al. Structural dynamics in the evolution of SARS-CoV-2
spike glycoprotein. Nat Commun. 2023;14(1):1421.
14. Chen C, Zhu R, Hodge EA, Diaz-Salinas MA, Nguyen A, Munro JB, et al.
hACE2-Induced Allosteric Activation in SARS-CoV versus SARS-CoV-2 Spike
Assemblies Revealed by Structural Dynamics. ACS Infect Dis.
2023;9(6):1180-9.
15. Costello SM, Shoemaker SR, Hobbs HT, Nguyen AW, Hsieh CL, Maynard
JA, et al. The SARS-CoV-2 spike reversibly samples an open-trimer
conformation exposing novel epitopes. Nat Struct Mol Biol.
2022;29(3):229-38.
16. Hanke L, Sheward DJ, Pankow A, Vidakovics LP, Karl V, Kim C, et al.
Multivariate mining of an alpaca immune repertoire identifies potent
cross-neutralizing SARS-CoV-2 nanobodies. Sci Adv. 2022;8(12):eabm0220.
17. Benhaim M, Lee KK, Guttman M. Tracking Higher Order Protein
Structure by Hydrogen-Deuterium Exchange Mass Spectrometry. Protein Pept
Lett. 2019;26(1):16-26.
18. Oganesyan I, Lento C, Wilson DJ. Contemporary hydrogen deuterium
exchange mass spectrometry. Methods. 2018;144:27-42.
19. Liu XR, Zhang MM, Gross ML. Mass Spectrometry-Based Protein
Footprinting for Higher-Order Structure Analysis: Fundamentals and
Applications. Chem Rev. 2020;120(10):4355-454.
20. Haque HME, Mantis NJ, Weis DD. High-Throughput Epitope Mapping by
Hydrogen Exchange-Mass Spectrometry. J Am Soc Mass Spectrom.
2023;34(1):123-7.
21. Seow J, Khan H, Rosa A, Calvaresi V, Graham C, Pickering S, et al. A
neutralizing epitope on the SD1 domain of SARS-CoV-2 spike targeted
following infection and vaccination. Cell Rep. 2022;40(8):111276.
22. Zhu S, Liuni P, Chen T, Houy C, Wilson DJ, James DA. Epitope
screening using Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS):
An accelerated workflow for evaluation of lead monoclonal antibodies.
Biotechnol J. 2022;17(2):e2100358.
23. Hamuro Y, Zhang T. High-Resolution HDX-MS of Cytochrome c Using
Pepsin/Fungal Protease Type XIII Mixed Bed Column. J Am Soc Mass
Spectrom. 2019;30(2):227-34.
24. Vorauer C, Wrigley MS, Rincon Pabon JP, Watson MJ, Mundorff CC, Weis
DD, et al. Rapid Assessment of Pepsin Column Activity for Reliable
HDX-MS Studies. J Am Soc Mass Spectrom. 2021;32(9):2386-90.
25. Mullahoo J, Zhang T, Clauser K, Carr SA, Jaffe JD, Papanastasiou M.
Dual protease type XIII/pepsin digestion offers superior resolution and
overlap for the analysis of histone tails by HX-MS. Methods.
2020;184:135-40.
26. Hamuro Y, Coales SJ. Optimization of Feasibility Stage for
Hydrogen/Deuterium Exchange Mass Spectrometry. J Am Soc Mass Spectrom.
2018;29(3):623-9.
27. Chau TH, Chernykh A, Kawahara R, Thaysen-Andersen M. Critical
considerations in N-glycoproteomics. Curr Opin Chem Biol.
2023;73:102272.
28. Xia Y, Ma Z, Qiu M, Guo B, Zhang Q, Jiang H, et al. N-glycosylation
shields Phytophthora sojae apoplastic effector PsXEG1 from a specific
host aspartic protease. Proc Natl Acad Sci U S A. 2020;117(44):27685-93.
29. Houde D, Arndt J, Domeier W, Berkowitz S, Engen JR. Characterization
of IgG1 Conformation and Conformational Dynamics by Hydrogen/Deuterium
Exchange Mass Spectrometry. Anal Chem. 2009;81(14):5966.
30. Zhang S, Li W, Lu H, Liu Y. Quantification of N-glycosylation site
occupancy status based on labeling/label-free strategies with LC-MS/MS.
Talanta. 2017;170:509-13.
31. Wu D, Struwe WB, Harvey DJ, Ferguson MAJ, Robinson CV. N-glycan
microheterogeneity regulates interactions of plasma proteins. Proc Natl
Acad Sci U S A. 2018;115(35):8763-8.
32. Zhao P, Praissman JL, Grant OC, Cai Y, Xiao T, Rosenbalm KE, et al.
Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human
ACE2 Receptor. Cell Host Microbe. 2020;28(4):586-601 e6.
33. Wang D, Baudys J, Bundy JL, Solano M, Keppel T, Barr JR.
Comprehensive Analysis of the Glycan Complement of SARS-CoV-2 Spike
Proteins Using Signature Ions-Triggered Electron-Transfer/Higher-Energy
Collisional Dissociation (EThcD) Mass Spectrometry. Anal Chem.
2020;92(21):14730-9.
34. Shajahan A, Pepi L, Kumar B, Murray N, Azadi P. Site Specific N- and
O-glycosylation mapping of the Spike Proteins of SARS-CoV-2 Variants of
Concern. Res Sq. 2022.
35. Zhu B, Chen Z, Shen J, Xu Y, Lan R, Sun S. Structural- and
Site-Specific N-Glycosylation Characterization of COVID-19 Virus Spike
with StrucGP. Anal Chem. 2022;94(36):12274-9.
36. Campos D, Girgis M, Sanda M. Site-specific glycosylation of
SARS-CoV-2: Big challenges in mass spectrometry analysis. Proteomics.
2022;22(15-16):e2100322.
37. Newby ML, Fogarty CA, Allen JD, Butler J, Fadda E, Crispin M.
Variations within the Glycan Shield of SARS-CoV-2 Impact Viral Spike
Dynamics. J Mol Biol. 2023;435(4):167928.
38. Krishnan S, Krishnan GP. N-Glycosylation Network Construction and
Analysis to Modify Glycans on the Spike (S) Glycoprotein of SARS-CoV-2.
Front Bioinform. 2021;1:667012.
39. Wang D, Zhou B, Keppel TR, Solano M, Baudys J, Goldstein J, et al.
N-glycosylation profiles of the SARS-CoV-2 spike D614G mutant and its
ancestral protein characterized by advanced mass spectrometry. Sci Rep.
2021;11(1):23561.
40. Saba J, Dutta S, Hemenway E, Viner R. Increasing the productivity of
glycopeptides analysis by using higher-energy collision
dissociation-accurate mass-product-dependent electron transfer
dissociation. Int J Proteomics. 2012;2012:560391.
41. Zhang J, Cai Y, Xiao T, Lu J, Peng H, Sterling SM, et al. Structural
impact on SARS-CoV-2 spike protein by D614G substitution. Science.
2021;372(6541):525-30.
42. Masson GR, Burke JE, Ahn NG, Anand GS, Borchers C, Brier S, et al.
Recommendations for performing, interpreting and reporting hydrogen
deuterium exchange mass spectrometry (HDX-MS) experiments. Nat Methods.
2019;16(7):595-602.
43. Li M, Zhu W, Zheng H, Zhang J. Efficient HCD-pd-EThcD approach for
N-glycan mapping of therapeutic antibodies at intact glycopeptide level.
Anal Chim Acta. 2022;1189:339232.
44. Shen Y, Xiao K, Tian Z. Site- and structure-specific
characterization of the human urinary N-glycoproteome with
site-determining and structure-diagnostic product ions. Rapid Commun
Mass Spectrom. 2021;35(1):e8952.
45. Sanda M, Benicky J, Goldman R. Low Collision Energy Fragmentation in
Structure-Specific Glycoproteomics Analysis. Anal Chem.
2020;92(12):8262-7.
46. Gong Y, Qin S, Dai L, Tian Z. The glycosylation in SARS-CoV-2 and
its receptor ACE2. Signal Transduct Target Ther. 2021;6(1):396.
47. Miyagi M, Nakazawa T. Determination of pKa values of individual
histidine residues in proteins using mass spectrometry. Anal Chem.
2008;80(17):6481-7.
48. Tran DT, Banerjee S, Alayash AI, Crumbliss AL, Fitzgerald MC. Slow
histidine H/D exchange protocol for thermodynamic analysis of protein
folding and stability using mass spectrometry. Anal Chem.
2012;84(3):1653-60.
49. Guttman M, Scian M, Lee KK. Tracking hydrogen/deuterium exchange at
glycan sites in glycoproteins by mass spectrometry. Anal Chem.
2011;83(19):7492-9.
50. Hatvany JB, Gallagher ES. Hydrogen/deuterium exchange for the
analysis of carbohydrates. Carbohydrate research. 2023;530:108859.
51. Rand KD, Zehl M, Jorgensen TJ. Measuring the hydrogen/deuterium
exchange of proteins at high spatial resolution by mass spectrometry:
overcoming gas-phase hydrogen/deuterium scrambling. Acc Chem Res.
2014;47(10):3018-27.
52. Wollenberg DTW, Pengelley S, Mouritsen JC, Suckau D, Jorgensen CI,
Jorgensen TJD. Avoiding H/D Scrambling with Minimal Ion Transmission
Loss for HDX-MS/MS-ETD Analysis on a High-Resolution Q-TOF Mass
Spectrometer. Anal Chem. 2020;92(11):7453-61.
53. Zehl M, Rand KD, Jensen ON, Jorgensen TJ. Electron transfer
dissociation facilitates the measurement of deuterium incorporation into
selectively labeled peptides with single residue resolution. J Am Chem
Soc. 2008;130(51):17453-9.
54. Mistarz UH, Bellina B, Jensen PF, Brown JM, Barran PE, Rand KD. UV
Photodissociation Mass Spectrometry Accurately Localize Sites of
Backbone Deuteration in Peptides. Anal Chem. 2018;90(2):1077-80.
55. Woo H, Park SJ, Choi YK, Park T, Tanveer M, Cao Y, et al. Developing
a Fully Glycosylated Full-Length SARS-CoV-2 Spike Protein Model in a
Viral Membrane. J Phys Chem B. 2020;124(33):7128-37.
56. Kumar P, Bhardwaj T, Garg N, Giri R. Microsecond simulations and CD
spectroscopy reveals the intrinsically disordered nature of SARS-CoV-2
spike-C-terminal cytoplasmic tail (residues 1242-1273) in isolation.
Virology. 2022;566:42-55.
57. Li F, Li W, Farzan M, Harrison SC. Structure of SARS coronavirus
spike receptor-binding domain complexed with receptor. Science.
2005;309(5742):1864-8.
58. Peterle D, DePice D, Wales TE, Engen JR. Increase the flow rate and
improve hydrogen deuterium exchange mass spectrometry. J Chromatogr A.
2023;1689:463742.
59. Peterle D, Wales TE, Engen JR. Simple and Fast Maximally Deuterated
Control (maxD) Preparation for Hydrogen-Deuterium Exchange Mass
Spectrometry Experiments. Anal Chem. 2022;94(28):10142-50.
60. Bai Y, Milne JS, Mayne L, Englander SW. Primary structure effects on
peptide group hydrogen exchange. Proteins. 1993;17(1):75-86.
61. Chetty PS, Mayne L, Lund-Katz S, Stranz D, Englander SW, Phillips
MC. Helical structure and stability in human apolipoprotein A-I by
hydrogen exchange and mass spectrometry. Proc Natl Acad Sci U S A.
2009;106(45):19005-10.
62. Gramlich M, Maier S, Kaiser PD, Traenkle B, Wagner TR, Voglmeir J,
et al. A Novel PNGase Rc for Improved Protein N-Deglycosylation in
Bioanalytics and Hydrogen-Deuterium Exchange Coupled With Mass
Spectrometry Epitope Mapping under Challenging Conditions. Anal Chem.
2022;94(27):9863-71.
63. Comamala G, Madsen JB, Voglmeir J, Du YM, Jensen PF, Osterlund EC,
et al. Deglycosylation by the Acidic Glycosidase PNGase H(+) Enables
Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass
Spectrometry. J Am Soc Mass Spectrom. 2020;31(11):2305-12.
64. Guo RR, Comamala G, Yang HH, Gramlich M, Du YM, Wang T, et al.
Discovery of Highly Active Recombinant PNGase H(+) Variants Through the
Rational Exploration of Unstudied Acidobacterial Genomes. Front Bioeng
Biotechnol. 2020;8:741.
65. Sztain T, Ahn SH, Bogetti AT, Casalino L, Goldsmith JA, Seitz E, et
al. A glycan gate controls opening of the SARS-CoV-2 spike protein. Nat
Chem. 2021;13(10):963-8.
66. Hsu YP, Frank M, Mukherjee D, Shchurik V, Makarov A, Mann BF.
Structural remodeling of SARS-CoV-2 spike protein glycans reveals the
regulatory roles in receptor-binding affinity. Glycobiology.
2023;33(2):126-37.
67. Casalino L, Gaieb Z, Goldsmith JA, Hjorth CK, Dommer AC, Harbison
AM, et al. Beyond Shielding: The Roles of Glycans in the SARS-CoV-2
Spike Protein. ACS Cent Sci. 2020;6(10):1722-34.