Identification of single amino acid substitutions (SAAS) in neuraminidase from influenza a virus (H1N1) via mass spectrometry analysis coupled with de novo peptide sequencing
peer-reviewed · Rapid Communications in Mass Spectrometry · 2016
| Date | 2016-08-01 |
| Type | peer-reviewed |
| Venue | Rapid Communications in Mass Spectrometry |
| Publisher | Wiley |
| Contribution | downstream-application |
| DOI | 10.1002/rcm.7650 |
| Citations (OpenAlex) | 1 |
| Venue 2-year citedness | 1.80 |
Abstract
Rationale Amino acid substitutions in the neuraminidase of the influenza virus are the main cause of the emergence of resistance to zanamivir or oseltamivir during seasonal influenza treatment; they are the result of non-synonymous mutations in the viral genome that can be successfully detected by polymer chain reaction (PCR)-based approaches. There is always an urgent need to detect variation in amino acid sequences directly at the protein level. Mass spectrometry coupled with de novo sequencing has been explored as an alternative and straightforward strategy for detecting amino acid substitutions, as well - this approach is the primary focus of the present study. Methods Influenza virus (A/Puerto Rico/8/1934 H1N1) propagated in embryonated chicken eggs was purified by ultracentrifugation, followed by PNGase F treatment. The deglycosylated virion was lysed and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel band corresponding to neuraminidase was picked up and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Results LC-MS/MS analyses, coupled with manual de novo sequencing, allowed the determination of three amino acid substitutions: R346K, S349 N, and S370I/L, in the neuraminidase from the influenza virus (A/Puerto Rico/8/1934 H1N1), which were located in three mutated peptides of the neuraminidase: YGNGVWIGK, TKNHSSR, and PNGWTETDI/LK, respectively. Conclusions We found that the amino acid substitutions in the proteins of RNA viruses (including influenza A virus) resulting from non-synonymous gene mutations can indeed be directly analyzed via mass spectrometry, and that manual interpretation of the MS/MS data may be beneficial. Copyright © 2016 John Wiley & Sons, Ltd.
Methods and tools
- Influenza neuraminidase substitutions by de novo sequencing: Detected three single amino acid substitutions in neuraminidase of influenza A/PR/8/34 H1N1 by LC-MS/MS with manual de novo sequencing of mutated peptides.
Methods it uses
- Manual MS/MS de novo interpretation: Protocol for manual de novo peptide sequencing from MS/MS spectra.