Influenza, the virus of the common flu, is associated with high infectivity and morbidity. This infection requires an obligate step of fusion of the viral coat with the endosomal membrane, subsequently allowing the release of the viral genome and proliferating machinery to the host cytosol. This fusion is driven by an acid-induced conformational change in the viral coat protein hemagglutinin (HA). To characterize this conformational change in relation with infectivity, we set out to develop a label-free, targeted approach to investigate HA fusion competence. Although monitoring the chemical activity of the protein backbone and side-chain residues has been widely applied to probe for protein conformation, coupling this assay to quantitative mass spectrometry has not been studied. Thus, we evaluated whether electrophilic amidination by thioimidate reagents coupled with targeted reaction monitoring mass spectrometry is an effective label-free approach to report the fusion-associated conformational switch in recombinant hemagglutinin. We identified peptides that are differentially amidinated between the pre- and post-fusion switch, and also validated that this difference is associated with the conformational switch. Additionally, it was noted that the quantitation maintains sensitivity when monitored in complex mixtures.

Protein profiling and pseudo-parallel reaction monitoring to monitor the fusion-associated conformational switch in hemagglutinin

2019 Fall National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.