Protein profiling and pseudo-parallel reaction monitoring to monitor a fusion-associated conformational change in hemagglutinin

Abstract

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 fusionassociated 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.

Profiling protein targets of cellular toxicant exposure

“…Even for proteins that defy two-state transitions, oxidation kinetics are a valid proxy for destabilization. 131 Electrophilic species have also been used for protein footprinting in biologically complex samples, including thioimidates, 126,[132][133][134] aldehydes, 135,136 maleimides, 137 and activated esters. 138 These probes are limited to nucleophilic residues such as cysteine, lysine, or histidine.…”