tetano
Editor, Senior Moderator
Anal Chem. 2017 Sep 18. doi: 10.1021/acs.analchem.7b03025. [Epub ahead of print]
[h=1]Characterization of Site-Specific glycosylation in Influenza A Virus Hemagglutinin produced by Spodoptera frugiperda insect cell line.[/h] Liu Y, Wu SL, Love KR, Hancock WS.
[h=3]Abstract[/h] Influenza hemagglutinin is a surface glycoprotein related to virus invasion and host immune system response. Understanding site specific glycosylation of hemagglutinin will increase our knowledge about virus evolution and can improve the design and quality of vaccines. In our study, we used glycoproteomic analysis based on multienzyme digestion followed by LC tandem MS analysis to determine the glycosylation of Influenza hemagglutinin (H1/A/California/04/2009) using the following steps: PNGaseF treatment combined with trypsin or pepsin digestion were used to determine the glycosites and glycan occupancy. Three enzymes, trypsin, AspN and pepsin, were used separately to generate suitable glycopeptides for on-line LC tandem MS analysis. The glycan structure of a given glycopeptide was determined by collision-induced dissociation MS/MS fragmentation and the peptide backbone information was provided by CID-MS3 fragmentation. With this approach 100% sequence coverage of the hemagglutinin sample was obtained. Six glycosylation sites fitting the sequon N-X-S/T were successfully confirmed and the glycan heterogeneity as well as the ratios of glycoforms were determined at each site.
PMID: 28921955 DOI: 10.1021/acs.analchem.7b03025
[h=1]Characterization of Site-Specific glycosylation in Influenza A Virus Hemagglutinin produced by Spodoptera frugiperda insect cell line.[/h] Liu Y, Wu SL, Love KR, Hancock WS.
[h=3]Abstract[/h] Influenza hemagglutinin is a surface glycoprotein related to virus invasion and host immune system response. Understanding site specific glycosylation of hemagglutinin will increase our knowledge about virus evolution and can improve the design and quality of vaccines. In our study, we used glycoproteomic analysis based on multienzyme digestion followed by LC tandem MS analysis to determine the glycosylation of Influenza hemagglutinin (H1/A/California/04/2009) using the following steps: PNGaseF treatment combined with trypsin or pepsin digestion were used to determine the glycosites and glycan occupancy. Three enzymes, trypsin, AspN and pepsin, were used separately to generate suitable glycopeptides for on-line LC tandem MS analysis. The glycan structure of a given glycopeptide was determined by collision-induced dissociation MS/MS fragmentation and the peptide backbone information was provided by CID-MS3 fragmentation. With this approach 100% sequence coverage of the hemagglutinin sample was obtained. Six glycosylation sites fitting the sequon N-X-S/T were successfully confirmed and the glycan heterogeneity as well as the ratios of glycoforms were determined at each site.
PMID: 28921955 DOI: 10.1021/acs.analchem.7b03025