tetano
Editor, Senior Moderator
Anal Bioanal Chem. 2019 Jun 28. doi: 10.1007/s00216-019-01921-z. [Epub ahead of print]
[h=1]Protein profiling and pseudo-parallel reaction monitoring to monitor a fusion-associated conformational change in hemagglutinin.[/h] Nguyen KK[SUP]1[/SUP], Thurmond S[SUP]2[/SUP], Hai R[SUP]3[/SUP], Genereux JC[SUP]4[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] Influenza infection requires viral escape from early endosomes into the cytosol, which is enabled by an acid-induced irreversible conformational transformation in the viral protein hemagglutinin. Despite the direct relationship between this conformational change and infectivity, label-free methods for characterizing this and other protein conformational changes in biological mixtures are limited. While the chemical reactivity of the protein backbone and side-chain residues is a proxy for protein conformation, coupling this reactivity to quantitative mass spectrometry is a challenge in complex environments. Herein, we evaluate whether electrophilic amidination coupled with pseudo-parallel reaction monitoring is an effective label-free approach to detect the fusion-associated conformational transformation in recombinant hemagglutinin (rHA). We identified rHA peptides that are differentially amidinated between the pre- and post-fusion states, and validated that this difference relies upon the fusion-associated conformational switch. We further demonstrate that we can distinguish the fusion profile in a matrix of digested cellular lysate. This fusion assay can be used to evaluate fusion competence for modified HA. Graphical abstract.
[h=4]KEYWORDS:[/h] Hemagglutinin; Mass spectrometry; Parallel reaction monitoring; Protein amidination; Protein conformation; Viral fusion
PMID: 31254054 DOI: 10.1007/s00216-019-01921-z
[h=1]Protein profiling and pseudo-parallel reaction monitoring to monitor a fusion-associated conformational change in hemagglutinin.[/h] Nguyen KK[SUP]1[/SUP], Thurmond S[SUP]2[/SUP], Hai R[SUP]3[/SUP], Genereux JC[SUP]4[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] Influenza infection requires viral escape from early endosomes into the cytosol, which is enabled by an acid-induced irreversible conformational transformation in the viral protein hemagglutinin. Despite the direct relationship between this conformational change and infectivity, label-free methods for characterizing this and other protein conformational changes in biological mixtures are limited. While the chemical reactivity of the protein backbone and side-chain residues is a proxy for protein conformation, coupling this reactivity to quantitative mass spectrometry is a challenge in complex environments. Herein, we evaluate whether electrophilic amidination coupled with pseudo-parallel reaction monitoring is an effective label-free approach to detect the fusion-associated conformational transformation in recombinant hemagglutinin (rHA). We identified rHA peptides that are differentially amidinated between the pre- and post-fusion states, and validated that this difference relies upon the fusion-associated conformational switch. We further demonstrate that we can distinguish the fusion profile in a matrix of digested cellular lysate. This fusion assay can be used to evaluate fusion competence for modified HA. Graphical abstract.
[h=4]KEYWORDS:[/h] Hemagglutinin; Mass spectrometry; Parallel reaction monitoring; Protein amidination; Protein conformation; Viral fusion
PMID: 31254054 DOI: 10.1007/s00216-019-01921-z