Glycoproteomic Analyses of Influenza A Viruses Using timsTOF Pro MS

Area of Science:

• Glycomics
• Mass Spectrometry
• Virology

Background:

• N-linked glycosylation on influenza hemagglutinin (HA) and neuraminidase (NA) impacts viral properties.
• Understanding site-specific glycoforms is crucial for influenza virus characterization and vaccine design.

Purpose of the Study:

• To develop and validate a glycoproteomic approach using timsTOF Pro mass spectrometry (MS) for analyzing site-specific glycosylation of influenza glycoproteins.
• To compare the performance of timsTOF Pro MS with Q Exactive HF MS for glycopeptide analysis.

Main Methods:

• Utilized timsTOF Pro mass spectrometry for glycoproteomic analysis of influenza HA glycoproteins.
• Quantified intact site-specific glycopeptides without hydrophilic interaction liquid chromatography (HILIC) enrichment.
• Employed ion mobility spectrometry (IMS) for glycopeptide analysis and differentiation of sialylation linkages.

Main Results:

• The timsTOF Pro MS method achieved comparable glycopeptide coverage and quantity to Q Exactive HF MS, while being more effective for low-abundance glycopeptides.
• Complex N-glycans were identified at specific sites (N22, N38, N240, N483) in H7N9 and H7N3 HA, with high-mannose glycans at N411.
• Distinct glycan types and quantities were observed between the H7N9 and H7N3 HA, and IMS differentiated sialylation linkages.

Conclusions:

• The timsTOF Pro MS method provides a robust and efficient approach for quantifying intact site-specific glycans on influenza glycoproteins without enrichment.
• This glycoproteomic strategy facilitates a deeper understanding of influenza virus heterogeneity.
• The method holds significant potential for advancing influenza vaccine development and production.