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Analytical and Bioanalytical Chemistry

, Volume 411, Issue 19, pp 4987–4998 | Cite as

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

  • Khanh K. Nguyen
  • Stephanie Thurmond
  • Rong HaiEmail author
  • Joseph C. GenereuxEmail author
Research Paper
Part of the following topical collections:
  1. Young Investigators in (Bio-)Analytical Chemistry

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 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

Keywords

Protein conformation Protein amidination Mass spectrometry Parallel reaction monitoring Viral fusion Hemagglutinin 

Notes

Funding information

This work was supported by a UCR Research and Economic Development SEED grant. Multiple reaction monitoring was performed on a TSQ Quantiva in the UCR Analytical Chemistry Instrumentation Facility, which was acquired from a NIH National Center for Research Resources grant 1S10RR022331. Molecular graphics and analyses performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with the support from NIH P41-GM103311.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2019_1921_MOESM1_ESM.pdf (2.3 mb)
ESM 1 (PDF 2.26 MB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of California RiversideRiversideUSA
  2. 2.Department of Plant Pathology & MicrobiologyUniversity of California RiversideRiversideUSA

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