Protein Sequon Peptide Glycan Glycan mass % base peak

39 KnVTVTHAQDILER/3 G6H3F 1850.69 10%

170 NVVWLIKKnSTYPTIKR/4 G2H5 1216.42 28%

181 RSYnNTNQE/3 G4H5F 1768.64 16%

G4H5FS 2059.73 88%

G4H5F 1768.64 12%

G6H6FS2 2919.04 18%

G2H6 1378.48 10%

G4H4F 1606.59 20%

CQTPMGAInSSMPFHNIHPLTIGECPKYVK/5 G6H4F 2012.75 12%

126 TFFLTQGALLNDKHSnGTVK/4 G6H3F3 2142.81 38%

215 CACVnGSCFTVMTDGPSNGQASHKIFK/4 G4H5FS 2059.73 18%

Hemagglutinin

Neuraminidase

302

CQTPMGAInSSMPFHNIHPLTIGECPK/5

CQTPMGAInSSMPFHNIHPLTIGECPK/4

Site-Specific Glycosylation Profiles for Recombinant Hemagglutinin and 

Neuraminidase from Different Sources

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

annually worldwide. Perhaps the most complex component of this virus is the 

N-linked glycan shield on its external proteins, hemagglutinin and 

neuraminidase. These proteins attach to host cells leading to infection and 

are the main antigens of interest for vaccine development and antiviral 

treatments. Their distributions of N-linked glycans at multiple sites are highly 

complex and serve to evade antibodies and perhaps serve other purposes. 

However, the accurate measurement of these site-specific glycan distributions 

and determination of their variability. However, the accurate measurement of 

these site-specific glycan distributions and determination of their variability

Introduction

Methodology

Results and Discussion

Conclusions

Zachary C. Goecker, Meghan C. Burke, Yi Liu, Yuri A. Mirokhin, Dmitrii V. Tchekhovskoi, Stephen E. Stein

• Variation due to vendor differences resulted in the lowest levels of library match (as low as 502 ± 218), whereas replicate, lot, and peptide variation resulted in high match scores (898 ± 73, 867 

± 101, and 870 ± 51 respectively).

• The most abundant glycans for BioVision and US Biological HA are G2H5, G5H3, and G4H3, whereas the most abundant glycans for Sino Biological are G2H5, G4H5FS, and G4H5F.

• Glycans that were present in hemagglutinin but not neuraminidase include G6H7FS3, G5H6FS2, and G5H6FS3. No glycans were identified in neuraminidase that were not also in hemagglutinin.

References and Disclaimers

Overview

• Glycopeptide abundance distribution spectra (GADS)

1 were created for influenza A recombinant glycoproteins hemagglutinin and neuraminidase (H5N1) to evaluate glycan variation.

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

• Glycan distribution was skewed toward higher mass (sialylated complex) glycans for Sino Biological and toward lower mass glycans (oligomannose and hybrid) for US Biological and BioVision.

Digital reprint

Recombinant proteins: 

Hemagglutinin strain A/Hong Kong/483/1997(H5N1) and neuraminidase 

strain A/Thailand/1(KAN-1)/2004(H5N1) were obtained from Sino Biological, 

BioVision, and US Biological. 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:

MS_Piano and NIST MS Search were used for data 

annotation and visualization. NIST developed 

software was used for glycopeptide and GADS library 

creation.

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

Replicate 1

Replicate 2

Replicate 3

Protein Sequon Match score

39 894 ± 84

170 935 ± 4

181 925 ± 76

302 893 ± 69

500 813 ± 73

68 869 ± 72

126 928 ± 36

215 929 ± 26

Hemagglutinin

Neuraminidase

Replicate variation Lot variation

Peptide variation

presents a significant analytical 

challenge. In 2020, Chang et al 

successfully compared glycans from 

hemagglutinin variants.2 This work 

attempts to develop methods for 

accurate and reproducible 

measurement of these distributions as 

well as for their detailed representation.

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.

2Chang, D., Hackett, W. E., Zhong, L., Wan, X. F., & Zaia, J. (2020). Measuring site-specific glycosylation similarity between influenza A virus variants with statistical certainty. Molecular & Cellular Proteomics, 19(9), 1533-1545.

3Jung, H. E., & Lee, H. K. (2020). Host protective immune responses against influenza A virus infection. Viruses, 12(5), 504.

Jung et al

2

SYnNTNQE

SYnNTNQEDLLVLWGIHHPNDAAEQTK

nNTNQEDLLVLW

IKRSYnNTNQEDLLV

0

100

200

300

400

500

600

700

800

900

1000

Hemagglutinin Neuraminidase

Library match score

BioVision Sino Biological

Trypsin + Lys-C Chymotrypsin

Trypsin + Asp-N Chymotrypsin + Glu-C

Trypsin + Glu-C alpha-Lytic

Trypsin + Chymotrypsin 

Figure 6. Protease combinations used

Table 1. Missing glycans between replicates

Figure 1. GADS for NA between three replicates

Table 2. Average match scores per protein glycosylation site

Figure 8. Inter-peptide match scores

Figure 7. GADS for different peptides from HA

0

200

400

600

800

1000

Hemagglutinin Neuraminidase

Library match score

Figure 3. GADS for HA between 2 lots

Figure 4. Inter-lot match scores

0

50

100

150

200

250

300

350

HA (Tryp+Glu-C) HA (Tryp+Asp-N) NA (Tryp+Glu-C) NA (Chymo)

Total Glycopeptides

Lot 1 Lot 2

Figure 5. Total glycopeptides between lots

Discussion:

To determine baseline variation, glycan 

profiles were compared between three 

biological replicates. Glycan distribution 

was similar between replicates (Fig 1), 

with few missing glycans between 

replicates (Table 1) and average match 

scores around 900/999 (Table 2).

Discussion:

Separate production lots were compared 

for both Sino Biological and BioVision. 

Glycan distribution patterns were similar 

(Fig 3), as well as the inter-lot match 

scores between identical peptides (Fig 

4). However, the total number of 

glycopeptides did fluctuate (Fig 5).

Discussion:

Glycopeptides from the same sequon

but which are cleaved at different 

amino acids (Fig 6) were compared via 

GADS (Fig 7) and inter-peptide library 

match scores (Fig 8). Glycan distribution 

was highly similar (>800) regardless of 

peptide cleavage.

Vendor variation

US Biological

BioVision

Sino Biological

0

100

200

300

400

500

600

700

800

900

1000

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

Library match score

US Biological BioVision Sino Biological

HA – sequon 39 - KnVTVTHAQDILER/4 G5H3 > G2H5 > G6H3 G6H3 > G5H3 > G2H5 G2H5 > G2H7 > G6H7FS2

HA – sequon 39 - KnVTVTHAQDILER/3 G2H5 > G5H3 > G3H3 G2H5 > G6H3 > G5H3 G2H5 > G5H6F > G2H7

HA – sequon 181 - SYnNTNQE/3 G4H3F > G5H3F > G5H4F G5H4FS > G4H5FS > G5H3FS G5H4F > G4H5F > G4H5FS

HA – sequon 181 - SYnNTNQE/2 G2H5 > G4H3F > G4H3 G4H3F > G4H3 > G2H5 G2H5 > G4H3F > G4H5F

HA – sequon 302 - CQTPMGAInSSMPFHNIHPLTIGECPK/4 G2H5 > G4H3 > G5H3 G2H5 > G4H3 > G5H3 G4H5FS > G4H5F > G5H6F

HA – sequon 302 - CQTPMGAInSSMPFHNIHPLTIGECPKYVK/5 G2H5 > G4H3 > G5H3 G2H5 > G4H3 > G5H3 G4H5FS > G4H5F > G2H5

HA – sequon 500 - SVKnGTYDYPQYSEE/3 G2H5 > G4H3 > G5H3 G4H3 > G2H5 > G5H3 G4H5FS > G2H5 > G5H6FS

NA – sequon 126 - TFFLTQGALLNDKHSnGTVK/4 G5H4F2 > G5H4F > G4H3F G4H3F > G5H4F2 > G5H4F G5H4F2 > G5H4F > G6H3F2

Figure 10. Top 3 abundant glycans for each glycopeptide

Figure 9. GADS for HA between three vendors

Figure 11. Library match scores between three vendors

Discussion:

Glycoproteins from BioVision, Sino 

Biological, and US Biological were 

compared in terms of glycan distribution 

patterns (Fig 9), most abundant glycans 

identified (Fig 10), and library matching 

between identical peptides (Fig 11).