Variation of Site-Specific Glycosylation Profiles for Influenza Glycoproteins from 

Recombinant Sources and Vaccines

Introduction

Methodology

Results and Discussion

Conclusions

Zachary C. Goecker, Meghan C. Burke, Yi Liu, Yuri A. Mirokhin, Sergey L. Sheetlin, Xiaoyu Yang, Dmitrii V. Tchekhovskoi, and Stephen E. Stein

• Inter-strain variation resulted in the lowest levels of library match (463 ± 130), followed by intra-strain variation (617 ± 151), and vendor variation (638 ± 218). However, variation between 

replicates (946 ± 38), lots (891 ± 57), and different protease combinations (870 ± 51) resulted in high match scores.

• Adjacent sequons may be isolated using EThcD fragmentation or non-specific cleavage using the protease subtilisin and most glycans are high-mannose in quadrivalent vaccines.

• Results herein demonstrate the advance of site-specific glycosylation methods in determining glycosylation patterns for influenza vaccines, which could be implemented in vaccine QC metrics.

References and Disclaimers

Overview

• Glycopeptide abundance distribution spectra (GADS)

1 were created for influenza A recombinant glycoproteins and commercial influenza monovalent and quadrivalent vaccines.

• Variation in glycan abundances and distribution was most pronounced between different strains and least pronounced between biological replicates of the same protein and same vendor.

• Adjacent sequons may be isolated via a non-specific protease or EThcD fragmentation and GADS for commercial influenza vaccines can be partially elucidated with current methods.

Digital reprint

Recombinant proteins: 

Nine hemagglutinin and neuraminidase strains were obtained from Sino 

Biological, BioVision, US Biological, and Creative Biomart. All proteins are 

from recombinant expression in HEK293 cell line. Multiple lots were ordered 

from these vendors.

Sample processing:

Purified recombinant protein was digested using 0.1% RapiGest surfactant in 

50 mM ABC, 20 mM DTT, and 55 mM IAA. Protein was digested using the 

following protease combinations: Trypsin+Glu-C, Trypsin+Lys-C, Trypsin+AspN, Trypsin+Chymotrypsin, Chymotrypsin, alpha-Lytic protease, and GluC+Chymotrypsin. Digests were purified and desalted using a MonoSpin

column procedure.

Instrumental analysis:

Digests were analyzed on a Thermo Scientific Fusion 

Lumos mass spectrometer using an optimized stepped 

HCD energy-ion trap fragmentation sequence.

Software:

NIST MS_Piano and NIST MS Search were used for 

data annotation and visualization. NIST developed 

software was used for glycopeptide and GADS library 

creation. GADS pseudo-spectra give the abundances 

of different glycans from the same peptide sequence.

Disclaimer: Certain commercial equipment, instruments, or materials are identified in this paper in order to specify the experimental procedure. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and 

Technology, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.

Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA

Variation in Glycosylation Patterns Isolating Adjacent Sequons

1Remoroza, 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.

2Jung, H. E., & Lee, H. K. (2020). Host protective immune responses against influenza A virus infection. Viruses, 12(5), 504.

Jung et al

2

Influenza Vaccines

EThcD Subtilisin non-specific protease Replicate Variation Lot Variation

Vendor Variation

Inter-strain Variation

Protease Variation

HEK293

Hen Egg

SYnNTNQE

SYnNTNQEDLLVLWGIHHPNDAAEQTK

nNTNQEDLLVLW

IKRSYnNTNQEDLLV

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

Biological

Technical

Influenza is a viral disease responsible for the death of ~500,000 people 

annually worldwide. As a result, vaccination against influenza is one of 

the most common immunizations, taken by about 50% of the U.S. 

population annually. On average, the effectiveness of the influenza 

vaccine is only 40%. One of the possible factors contributing this low 

efficacy is the variation of the glycan distributions on the external 

proteins, hemagglutinin and neuraminidase. The accurate 

measurement of these site-specific glycan distributions and

determination of their 

variability presents a significant 

analytical challenge. This work 

develops methods for both 

accurate and reproducible 

measurements of these 

distributions to reduce 

uncertainty in vaccine 

development.

Figure 7. Tandem mass spectrum of glycopeptide containing 

the adjacent sequon. One sequon is unoccupied as shown by 

the z ion series. However, not all z ions in the series are 

present.

Figure 8. The non-specific protease subtilisin may be used 

to cleave between two adjacent asparagines, regardless 

of glycan occupancy. GADS are obtained which contain 

many supporting tandem spectra.

Intra-strain Variation

Figure 10. Sequence alignment of the 

four hemagglutinin strains found in 

the annual quadrivalent flu shot. 

Many sequons share a sequence with 

other strains, both in confined (red) 

and expansive (green) regions.

Figure 9. Hemagglutinin and 

neuraminidase are two glycoproteins 

found in influenza vaccines.

Figure 11. Typical GADS for recombinant 

hemagglutinin from HEK293 cell line are 

complex, whereas typical GADS from 

hemagglutinin from hen eggs are highmannose.

Average Match Score: 617 ± 151

0

200

400

600

800

1000

Hemagglutinin Neuraminidase

Figure 2. Inter-lot match scores

Figure 1. Biological and technical replicate match scores

0

100

200

300

400

500

600

700

800

900

1000

BioVision - Sino Bio BioVision - US Bio Sino Bio - US Bio

Figure 3. Library match scores between three vendors

Average match: 819 ± 106

Figure 4. GADS from different peptides of the same sequon

...NNST...

...NNTT...

...NNTS...

...NNSS...

ANNSTDTVDTVL

HANNSTDTVDTVL

HANNSTDTVDTV

nSTDTVDTVL } nSTDTVDTVLEK

Adjacent 

sequon 

motifs

# of associated spectra = 140

Figure 5. GADS comparison between nine influenza strains

Figure 6. Match scores between different glycosylation 

sites within the same protein

Sequon 79

Sequon 54

Sequon 38

Average Match Score: 463 ± 130

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