title: Identification of single amino acid substitutions (SAAS) in neuraminidase from influenza a virus (H1N1) via mass spectrometry analysis coupled with de novo peptide sequencing.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun, 130062, China.|Central Laboratory, Second Hospital, Jilin University, Changchun, 130041, China.
authors.forename: Qisheng
authors.initials: Q
authors.lastname: Peng
authors.affiliation: Central Laboratory, Second Hospital, Jilin University, Changchun, 130041, China.
authors.forename: Zijian
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Center for Disease Control and Prevention, Changchun, 130025, Jilin, China.
authors.forename: Donglin
authors.initials: D
authors.lastname: Wu
authors.affiliation: Tumor Hospital of Jilin Province, Changchun, 130022, China.
authors.forename: Xiaoou
authors.initials: X
authors.lastname: Li
authors.affiliation: Tumor Hospital of Jilin Province, Changchun, 130022, China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Liu
authors.affiliation: Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun, 130062, China.
authors.forename: Wanchun
authors.initials: W
authors.lastname: Sun
authors.affiliation: Central Laboratory, Second Hospital, Jilin University, Changchun, 130041, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Liu
mesh_terms: D019943:Amino Acid Substitution; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D053118:Influenza A Virus, H1N1 Subtype / Q000201:enzymology* / Q000235:genetics; D009439:Neuraminidase / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D009439:Neuraminidase