Material Measurement Laboratory

Variation of Site-Specific Glycosylation 

Profiles for Influenza Glycoproteins 

from Different Sources

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

Dmitrii Tchekhovskoi, Xiaoyu Yang, and Stephen Stein

BMD Staff Seminar

May 17, 2022
Material Measurement Laboratory

Why Glycopeptides at NIST?

• Tandem libraries are growing.

• Glycopeptides prominent in biologics.

• Measurement of glycosylation is difficult and hard to reproduce.

• Variation in glycosylation pattern unknown. How reproducible?

NISTMab
Material Measurement Laboratory

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. 1
Material Measurement Laboratory

Influenza Vaccine

• Quadrivalent: 4 strains

• 2 Influenza A strains

• 2 Influenza B strains

• Embryonated chicken eggs

• Inactivated by rupturing membrane

• Adjuvants

Influenza A (H3N2) Influenza A (H1N1)

Influenza B (Victoria) Influenza B (Yamagata)
Material Measurement Laboratory

Quality Control of Influenza Vaccines

Academic 

Research

Preclinical 

Testing Phase I Phase II Phase III Licensure

Clinical QC

• Effectiveness

• Antibody production

• Safety

• Use for at-risk populations

Pre-Clinical QC

• Characterization of 

physicochemical properties

• Potency

• Stability

• Batch variability

• Immunogenicity

• Toxicity

Investigational New 

Drug (IND) application
Material Measurement Laboratory

Why Glycosylation Matters For Vaccines

2

3

...THLNFKYPAL...

...THLNFTYPAL...

Sequon motif

NXT/S X≠P
Material Measurement Laboratory

Methods

HEK293

Reduction

Alkylation

Digestion

Purification

HCD - 15%, 25%, 35% NCE 

Oxonium ions

Peptide backbone ions

Glycopeptide ions
Material Measurement Laboratory

Methods – Data Processing

NIST 

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

Glycopeptide Abundance Distribution Spectrum
Material Measurement Laboratory

Variation in glycosylation profile - Replicate

Biological Replicate Technical Replicate

Protein Sequon Average match score

39 980 ± 2

170 913 ± 60

181 968 ± 16

302 884 ± 92

500 884 ± 27

68 973 ± 6

126 945 ± 21

215 959 ± 11

Total 939 ± 62

Hemagglutinin

Neuraminidase

Protein Sequon Average match score

39 963

170 967

181 939 ± 40

302 946 ± 44

500 926 ± 27

68 939 ± 14

126 961 ± 24

215 927 ± 68

Total 946 ± 38

Hemagglutinin

Neuraminidase
Material Measurement Laboratory

Variation in glycosylation profile - Lot

Batch Variation Lot Variation

Protein Sequon Average match score

68 903 ± 39

126 852 ± 72

215 910 ± 48

Total 891 ± 57

Neuraminidase

Protein Sequon Average match score

39 889 ± 75

170 843 ± 38

181 942 ± 19

302 909 ± 45

500 872 ± 69

Total 897 ± 57

Hemagglutinin
Material Measurement Laboratory

Variation in glycosylation profile - Proteases

• Trypsin + Lys-C

• Trypsin + Glu-C

• Trypsin + Chymotrypsin

• Chymotrypsin + Glu-C

• Chymotrypsin

• Alphalytic protease

SYnNTNQE

SYnNTNQEDLLVLWGIHHPNDAAEQTK

nNTNQEDLLVLW

IKRSYnNTNQEDLLV

Average match: 819 ± 106
Material Measurement Laboratory

Variation in glycosylation profile - Vendor

Vendors

• BioVision

• Sino Biological

• US Biological

Strains

• HA (A/Hong Kong/483/1997) H5N1

• NA (A/Thailand/1(KAN-1)/2004) H5N1

0

100

200

300

400

500

600

700

800

900

1000

Vendor 1 - Vendor 2 Vendor 1 - Vendor 3 Vendor 2 - Vendor 3

Cumulative Glycopeptide Match Scores

Average match score: 638 ± 218
Material Measurement Laboratory

Variation in glycosylation profile – Same Strain

Average Match Score: 617 ± 151

Sequon 79

Sequon 54

Sequon 38
Material Measurement Laboratory

Variation in glycosylation profile – Different Strains

Protein Strain Subtype Number of sequons Protein mass* (kDa)

HA A/California/04/2009 H1N1 8 63

N A A/California/04/2009 H1N1 8 52

HA A/Hong Kong/483/1997 H5N1 8 64

HA A/Hong Kong/485197/2014 H3N2 13 64

HA A/Japan/305/1957 H2N2 8 63

HA A/New Caledonia/20/1999 H1N1 10 63

N A A/Arizona/13/2008 H1N1 9 52

N A A/Netherlands/219/2003 H7N7 11 52

N A A/Thailand/1(KAN-1)/2004 H5N1 3 49

*Unglycosylated

Average Match Score: 

463 ± 130
Material Measurement Laboratory

Summary of Variation

0

100

200

300

400

500

600

700

800

900

1000

Dot Product

Sources of Variation in GADS • Glycosylation of replicates is 

most similar and between 

strains is least similar.

• More deviation associated with 

lower match scores.

• “Random” is measured between 

different proteins from different 

vendors. (A1AG vs HA)
Material Measurement Laboratory

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...
Material Measurement Laboratory

Isolation of Adjacent Sequons - EThcD

• ETD (Electron Transfer Dissociation)

• HCD (High-Energy Collisional Dissociation)

HCD

ETD
Material Measurement Laboratory

Isolation of Adjacent Sequons - EThcD

• ETD (Electron Transfer Dissociation)

• HCD (High-Energy Collisional Dissociation)

GSYNNTSGEQMLIIWGVHHPIDETEQR HCD

ETD
Material Measurement Laboratory

Isolation of Adjacent Sequons - Subtilisin

• Bacillus subtilis – extracellular serine endopeptidases

nSTEQVDTIME 

HANNSTEQVDTIMEK 

DQICIGYHAn ??? 
Material Measurement Laboratory

Next Steps: Influenza Vaccines

• Afluria Quadrivalent

• Split virion

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

• Thimerosal

• Mercury

• Sodium Chloride

• Sodium Phosphate

• Potassium Phosphate

• Potassium Chloride

• Calcium Chloride

• Sodium Taurodeoxycholate

• Ovalbumin

• Sucrose.........

• 67 sequons within all HA and NA proteins

+
Material Measurement Laboratory

Influenza Vaccines – Challenges and Solutions
Material Measurement Laboratory

Afluria GADS

• ~8 of the 40 sequons for the four hemagglutinin proteins identified.

• The GADS are majority high-mannose. 

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

abundance of NA on the virus.
Material Measurement Laboratory

Afluria GADS

• ~8 of the 40 sequons for the four hemagglutinin proteins identified.

• The GADS are majority high-mannose. 

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

abundance of NA on the virus.
Material Measurement Laboratory

Conclusions

• Glycosylation of replicates is most similar and between strains is least 

similar.

• Adjacent sequons may be isolated using EThcD fragmentation or nonspecific cleavage using the protease subtilisin.

• Most glycans are high-mannose in egg-based quadrivalent vaccines.

• Next steps: 

• Optimize methods for influenza vaccine digestion.

• Build reference MS2 and GADS libraries

• Apply methods to other viral glycoproteins (HIV, Herpesvirus, Ebola...)
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
Material Measurement Laboratory

References

1Jung, H. E., & Lee, H. K. (2020). Host protective immune responses against influenza A virus 

infection. Viruses, 12(5), 504.

2Remoroza, 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.

3https://www.cdc.gov/flu/vaccines-work/effectiveness-studies.htm

4Zost, 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.
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

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