Material Measurement Laboratory

Measuring Variation in Glycosylation 

Among Influenza Vaccine Glycoproteins

Zachary Goecker, Meghan Burke, Concepcion Remoroza, Yi Liu, Yuri Mirokhin, Sergey Sheetlin,

Dmitrii Tchekhovskoi, Xiaoyu Yang, and Stephen Stein

BMD Staff Seminar

Jan 31, 2023
Material Measurement Laboratory

BMD Seminar Déjà Vu?
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Influenza Virus

• 10 proteins

• Hemagglutinin (HA) and 

neuraminidase (NA) 

transmembrane proteins.

• Diversity of strains arise 

through two mechanisms: 

point mutations in the viral 

genome or reassortment 

between two co-circulating 

strains. Jung et al., 2020
Material Measurement Laboratory

Why Glycosylation Matters For Vaccines

...THLNFKYPAL...

...THLNFTYPAL...

Sequon motif

NXT/S X≠P

Zost et al., 2017 www.cdc.gov
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Site-Specific Glycosylation

HCD - 15%, 25%, 35% NCE 

Oxonium ions

Peptide backbone ions

Glycopeptide ions
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Visualizing Glycan Distributions

Watanabe et al

Chang et al

1. Glycopeptide search using 

Byonic software

2. Tandem library creation and 

spectral validation via 

create_glycopeptide_lib.exe and 

MS_Piano.exe.

3. GADS creation using makegads.exe
Material Measurement Laboratory

Glycopeptide Abundance Distribution Spectrum

Visualizing Glycan Distributions

1. Glycopeptide search using 

Byonic software

2. Tandem library creation and 

spectral validation via 

create_glycopeptide_lib.exe and 

MS_Piano.exe.

3. GADS creation using makegads.exe
Material Measurement Laboratory

Data Validation

• Hyperscore

• Retention time

• Contingent ion trap scan

• Oxonium ions

• Number of instances

• Glycopeptide (Y) ion series

• MS1 XIC

• MS1 Isotopic envelope

• MS2 purity

• Precursor mass overlap
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Recombinant Proteins
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Review - Variation in glycosylation profile

Inter-Protein
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Review - Variation in glycosylation profile

Inter-Protein
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Variation From Different Protease Digestions

Protease Combination Amino Acid Cleavage Sites

Trypsin + Lys-C KR

Trypsin + Glu-C KRED

Trypsin + Chymotrypsin KRFWYL

Chymotrypsin + Glu-C FWYLED

Chymotrypsin FWYL

Alpha-lytic TASV

RLSTHNVINAEDAPGGPYEIGTSGSCPNITNGNGFFATMAWAVPKL
Material Measurement Laboratory

Variation From Different Protease Digestions

Protease Combination Amino Acid Cleavage Sites

Trypsin + Lys-C KR

Trypsin + Glu-C KRED

Trypsin + Chymotrypsin KRFWYL

Chymotrypsin + Glu-C FWYLED

Chymotrypsin FWYL

Alpha-lytic TASV

RLSTHNVINAEDAPNGTYEIGTSGSCPNITNGNGFFATMAWAVPKL
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Variation From Different Protease Digestions
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Variation From Different Strains
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Conserved Regions Have Conserved Glycosylation Distribution

Jang et al., 2014
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Variation from different proteins

Inter-Protein

Hemagglutinin A1AG
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Sars-CoV-2 Spike protein

Influenza Hemagglutinin

Inter-Protein Comparison
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Review - Isolation of Adjacent Sequons

• Site-specific glycosylation methods require a single sequon per 

peptide. Different proteases are used to maximize isolation.

• Influenza, HIV, Ebola, Herpesvirus, and MERS have adjacent sequons

NXT/S X≠P

NNSS, NNTT, NNST, NNTS

...PIDETEQGSYNNTSGEQMLIIWGVHHR...
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Isolation of Adjacent Sequons - Subtilisin

• Bacillus subtilis – extracellular serine endopeptidases

NSTEQVDTIME 

HANNSTEQVDTIMEK 

DQICIGYHAN ??? 
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Isolation of Adjacent Sequons - Subtilisin

• Bacillus subtilis – extracellular serine endopeptidases

• Three more proteases from Bacillus subtilis were obtained

• Esperase

• Savinase

• Neutrase
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Influenza Vaccines

• Contents

• Split virion

• 60 mg HA per dose (15 mg per strain)

• Thimerosal

• Mercury

• Sodium Chloride

• Sodium Phosphate

• Sodium Taurodeoxycholate

• Ovalbumin

• Sucrose.........

• Afluria Quadrivalent

• 3 Monovalent vaccines from NIBSC

• 3 Monovalent vaccines from Creative Biomart

+
Material Measurement Laboratory

Quadrivalent Vaccine Challenges
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Afluria GADS

• 8→23 of the 40 sequons for the four hemagglutinin proteins

identified. 27 are non-overlapping

• Three major GADS classes: high mannose, G4F complex, G5F complex

• No GADS were identified for NA proteins, probably due to a lower 

abundance of NA on the virus.
Material Measurement Laboratory

Afluria GADS

• 8→23 of the 40 sequons for the four hemagglutinin proteins

identified. 27 are non-overlapping

• Three major GADS classes: high mannose, G4F complex, G5F complex

• No GADS were identified for NA proteins, probably due to a lower 

abundance of NA on the virus.
Material Measurement Laboratory

Afluria GADS

• 8→23 of the 40 sequons for the four hemagglutinin proteins

identified. 27 are non-overlapping

• Three major GADS classes: high mannose, G4F complex, G5F complex

• No GADS were identified for NA proteins, probably due to a lower 

abundance of NA on the virus.
Material Measurement Laboratory

Monovalent vaccine GADS

• 90% sequons detected

• Three major GADS classes: high mannose, G4/G5, G4/G5 + high 

mannose

• Again, no major GADS identified for NA.

G4H5F

G5H6F

G6H7F

G7H8F

G8H9F

G6H7F4

G6H7F3

G5H6F2 G6H7F2

G5H6F3

G5H5F

G4H5F3
Material Measurement Laboratory

Open Search for Vaccine Modifications

Mass shift (Da) Modification

15.99 Oxidation

28.03 Ethylation

42.00 Acetylation

72.02 Carboxyethyl

113.08 Acetyl Deoxyhypusine

145.03 ?

200.11 ?

226.16 ?

Formalin

Formalin

B-Propiolactone
Material Measurement Laboratory

Next Steps

• Three more commercial vaccines were purchased for the 2022-2023 

flu season

• Flublok – Recombinant vaccine 

• Afluria – Egg based vaccine

• Flucelvax – Cell culture based vaccine

• How does glycosylation change... year to year

... strain to strain

... between sources
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Next Steps

• Characterize forensic evidence using glycosylation.

• Classification of sperm and egg glycosylation for 

determining likelihood of fertilization.
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Conclusions

• Glycosylation profiles between replicates is most similar and between 

different proteins is least similar.

• Homologous sequence regions between different influenza strains 

have similar glycosylation distribution compared to non-conserved 

regions.

• Most glycans are high-mannose or mono-fucoylated complex in eggbased quadrivalent vaccines.

• Next steps: 

• Assess more subtilisin proteases ability to isolate adjacent sequons

• Digest 2022-2023 vaccines and compare glycan distributions

• Build reference MS2 and GADS libraries
Material Measurement Laboratory

Acknowledgments

Software 

• Yuri Mirokhin

• Sergey Sheetlin

• Dmitrii Tchekhovskoi

• Xiaoyu Yang

• Guanghui Wang

• Stephen Stein

Lab mentoring 

• Yi Liu

• Jane Zhang

Data acquisition/analysis 

• Meghan Burke Harris

• Connie Remoroza

Administrative support 

• Heather Blache

• Sarah Huber

• Bill Wallace

Post-doc Adviser 

• Stephen Stein
Material Measurement Laboratory

References

Jung, H. E., & Lee, H. K. (2020). Host protective immune responses against influenza A virus 

infection. Viruses, 12(5), 504.

Remoroza, C. A., Burke, M. C., Liu, Y., Mirokhin, Y. A., Tchekhovskoi, D. V., Yang, X., & Stein, S. E. (2021). 

Representing and Comparing Site-Specific Glycan Abundance Distributions of Glycoproteins. Journal of Proteome 

Research, 20(9), 4475-4486.

https://www.cdc.gov/flu/vaccines-work/effectiveness-studies.htm

Zost, S. J., Parkhouse, K., Gumina, M. E., Kim, K., Perez, S. D., Wilson, P. C., ... & Hensley, S. E. (2017). 

Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by eggadapted vaccine strains. Proceedings of the National Academy of Sciences, 114(47), 12578-12583.

Watanabe, Y., Allen, J. D., Wrapp, D., McLellan, J. S., & Crispin, M. (2020). Site-specific glycan analysis of the 

SARS-CoV-2 spike. Science, 369(6501), 330-333.

Chang, D., & Zaia, J. (2019). Why glycosylation matters in building a better flu vaccine. Molecular & Cellular 

Proteomics, 18(12), 2348-2358.

Jang, Y. H., & Seong, B. L. (2014). Options and obstacles for designing a universal influenza vaccine. Viruses, 

6(8), 3159-3180.
Material Measurement Laboratory

Questions?

zachary.goecker@nist.gov
Material Measurement Laboratory

Supplemental Slides

Dot Product

Cosine of the angle between spectra 

represented as vectors

(Σ WLWU)

2

Σ WL

2 Σ WU2

WL = Weighted intensity of library

WU = Weighted intensity of unknown
Material Measurement Laboratory

Supplemental Slides
Material Measurement Laboratory

Supplemental Slides

Glycobiology

• In viruses

• Immune evasion

• Host cell attachment

• In mammalian cells

• Protein folding

• Protein stabilization

• Communication

• Function
Material Measurement Laboratory

Glycan distribution was skewed toward higher mass (sialylated complex) glycans for 

vendor 2 and toward lower mass glycans (oligomannose and hybrid) for vendor 1 

and vendor 3.

Vendor Comparison

Supplemental Slides

Vendor 2
Material Measurement Laboratory

Supplemental Slides

EThcD Stepped HCD

Peptide bond

Glycosidic bond

N-glycosidic bond

Fluoranthene