Material Measurement Laboratory

Developing Reproducible Methods in 

Site-Specific Glycosylation Analysis

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

Guanghui Wang, Dmitrii Tchekhovskoi, Xiaoyu Yang, and Stephen Stein

WBMSDG Seminar

Feb 27, 2023
Material Measurement Laboratory

NIST Mass Spectrometry Data Center
Material Measurement Laboratory

Mass Spectrometry of Glycoproteins

Analysis methods

• NMR

• Glycan structure

• Segmental dynamics

• Mass Spectrometry

• Released Glycan

• Intact glycopeptides (site-specific)

Human Milk Proteins

• Lactoferrin

• IgA1, IgA2, IgJ

• PIGR

• Tenascin 

Virus Proteins

• Influenza HA + NA

• Sars-CoV-2 Spike

Plasma Proteins

• Transferrin

• Haptoglobin

• 30+ other serum glycoproteins

Other Relevant Proteins

• Prostate specific antigen

Biologics (mAb)

• Repatha

• Remicade

• Embrel

• 3+ other mAb
Material Measurement Laboratory

Site-Specific Glycosylation

HCD - 15%, 25%, 35% NCE 

Oxonium ions

Peptide backbone ions

Glycopeptide ions
Material Measurement Laboratory

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
Material Measurement Laboratory

XIC Browser
Material Measurement Laboratory

Purified proteins

Human milk (NIST SRM 1953)

Interlaboratory Studies

Objectives of the study

❑ To build a searchable MS library format of MS1 data to 

compare relative abundances

❑ To develop a Tandem MS Library of annotated human 

milk N-glycopeptides

Milk Glycoproteins
Material Measurement Laboratory

(tenascin) KFDRYRLnYSLPT/+3+4 Class:HiMan=0.703,Hybrid=0.070,Complex=0.043,Fucose=0.000,Sialyl=0.051 Sequon:1018 File:Tenascin_3_urea_NLumos_2020-09-16_alphalyt_120_NM_HS

1200 1280 1360 1440 1520 1600 1680 1760 1840 1920 2000 2080 2160 2240 2320 2400 2480 2560 2640 2720

0

50

100

G2H5

G3H4

G2H6

G3H5

G2H7

G3H6G4H5G5H4

G2H8 G4H5S

Site-specific Glycosylation AnalysisGADS of Tenascin

NIST MASS SPECTROMETRY DATA CENTER

(tenascin) EVGWDGLRLnWT/+2+3+4 Class:HiMan=0.000,Hybrid=0.000,Complex=0.964,Fucose=0.997,Sialyl=0.790 Sequon:1093 File:TNC_06_urea_L1_2021-08-04_380-2000_1

1200 1280 1360 1440 1520 1600 1680 1760 1840 1920 2000 2080 2160 2240 2320 2400 2480 2560 2640 2720

0

50

100

G3H4F G4H5

G4H5F

G4H5S

G4H5FS

G5H4FS

G5H6F

G5H6FSo>4

G4H5FS2

G5H6FS

G6H7F

G5H7FS

G5H6FS2

G6H5FS2

Relative Abundance
Material Measurement Laboratory

IgA1

144

340

NIST MASS SPECTROMETRY DATA CENTER

Measurement Reproducibility and VariabilityGADS of IgA1

30% and IT 35%

Stepped 15 30 40% NCE

40% NCE

Relative Abundance
NIST MASS SPECTROMETRY DATA CENTER Material Measurement Laboratory

Lactoferrin

156 497 642

GADS of Lactoferrin (Human Milk) Relative Abundance
Utrecht University (PXD014917) NIST MASS SPECTROMETRY DATA CENTER Material Measurement Laboratory

Human milk (Interlab)-Application of GADS

Query GADS

N156

SRM 1953-NIST

Zhu et al, J. Nutrition 2021

N156

Milk Donor 1-Utrecht U.
Material Measurement Laboratory

Lessons learned from Kallikrein 

Metaheterogeneity

(Yi Liu) 
Material Measurement Laboratory

Why Glycosylation Matters For Vaccines

...THLNFKYPAL...

...THLNFTYPAL...

Sequon motif

NXT/S X≠P

Zost et al., 2017 www.cdc.gov
Material Measurement Laboratory

Site-specific glycan analysis of the SARS-CoV-2 spike, Volume: 369, Issue: 6501, Pages: 330-333, DOI: (10.1126/science.abb9983) 

SARS CoV-2 Spike Protein
Material Measurement Laboratory

11 Sources of Recombinant Spike

Source Sequence Cells Furin Pro Substitution Mutations Tag

A 16-1213 HEK293 RAAA T4, 10xHis

B 16-1213 HEK293 RAAA F817, A892, A899, 

A942, K986, K87

T4, 10xHis

C 16-1213 HEK293 RAAA His

D 1-1273 HEK293 Expi GSAG K986, V987 Rho 1D4

E 1-1273 HEK293 Expi GSAG K986, V987 del 69-70 & 144, 

N501Y, A570 D, 

D614G, P681H, T716I

Rho 1D4

F 15-1208 HEK293 GSAS K986, K987 6xHis

G 1-1208 CHOExpress GSAS F817, A892, A899, 

A942, K986, V987

del 69-70 & 144-145, 

N501Y, A570D, 

D614G, P681H

8xHis

H 1-1208 CHOExpress GSAS K986, K987 8xHis

I 15-1208 HEK293 GSAS K986, K987 His

J 16-1188 HEK RAAA T4, His+Avi

K 16-1213 HEK293 RAAA T4, 10xHis
Material Measurement Laboratory

GADS Are Reproducible
Material Measurement Laboratory

Glycosylation Sites Vary in Abundance
Material Measurement Laboratory

Similar Preparations Produce Similar Glycan Profiles
Material Measurement Laboratory
Material Measurement Laboratory

Influenza Virus

• 10 proteins

• Hemagglutinin (HA) and 

neuraminidase (NA) 

transmembrane proteins.

• Diversity of strains arise 

through point mutations or 

reassortment.

Jung et al., 2020
Material Measurement Laboratory

Variation in glycosylation profile

Inter-Protein
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

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
Material Measurement Laboratory

Variation From Different Protease Digestions
Material Measurement Laboratory

Variation From Different Strains
Material Measurement Laboratory
Material Measurement Laboratory

Conserved Regions Have Conserved Glycosylation Distribution

Jang et al., 2014
Material Measurement Laboratory

Sars-CoV-2 Spike protein

Influenza Hemagglutinin

Inter-Protein Comparison
Material Measurement Laboratory

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
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

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 Afluria Vaccine Modifications

Mass shift (Da) Modification

-2.01 Val → Pro

14.01 Methylation

15.99 Oxidation

52.91 Fe(III)

57.02 +Gly

72.02 Carboxyethyl

113.08 +Leu/Ile

128.09 +Lys

129.04 +Glu

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
Material Measurement Laboratory

Conclusions

• Milk glycoproteins exhibit diverse GADS between sites, but are 

reproducible between labs and MS methods.

• Recombinant Sars-CoV-2 spike protein is highly reproducible in 

glycosylation distribution between vendor and processing method, 

but is very different in glycan abundance depending on the site.

• Hemagglutinin replicates are most similar in glycosylation profile and 

digests from different proteins are least similar in glycosylation 

profile.

• Homologous sequence regions between different influenza strains 

have similar glycosylation distribution compared to non-conserved 

regions.
Material Measurement Laboratory

Acknowledgments

Steve Stein

Bill Wallace

Meghan Burke

Connie Remoroza

Yi Liu

Yuri Mirokhin

Sergey Sheetlin

Dmitrii Tchekhovskoi

Guanghui Wang

Sara Yang
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

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 - Subtilisin

• Bacillus subtilis – extracellular serine endopeptidases

NSTEQVDTIME 

HANNSTEQVDTIMEK 

DQICIGYHAN ??? 
Material Measurement Laboratory

Isolation of Adjacent Sequons - Subtilisin

• Bacillus subtilis – extracellular serine endopeptidases

• Three more proteases from Bacillus subtilis were obtained

• Esperase

• Savinase

• Neutrase
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