Serotype classification and characterisation of the rotavirus SA11 VP6 protein using mass spectrometry and two-dimensional gel electrophoresis

Kerry R. Emslie*, Mark P. Molloy, Celia R M Barardi, Daniel Jardine, Marc R. Wilkins, A. Richard Bellamy, Keith L. Williams

*Corresponding author for this work

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

VP6, which makes up the inner capsid of rotavirus, is the major structural protein of this virus. Whilst VP6 has been sequenced at the DNA level in several rotavirus strains, there has been less effort to characterise the protein at the amino acid level. This paper reports the use of peptide mass fingerprinting and post-source decay fragmentation studies using MALDI-TOF and electrospray ionisation mass spectrometry to identify and characterise, in detail, the VP6 protein. We show that mass spectrometric analysis of VP6 peptides successfully distinguished SA11 from other rotavirus serotypes, and identify unique peptides that can be used for serotypic differentiation. For VP6 characterisation, the ExPASy FindMod tool was used to predict post-translational modifications on the protein. Analysis of trypsin and AspN digests predicted that the N-terminal methionine of VP6 was acetylated and this was confirmed using post source decay and electrospray ionisation mass spectrometry-mass spectrometry. An asparagine residue (aa107), which is followed by a glycine residue, was shown to undergo partial deamidation to aspartic acid. VP6 has two additional asparagine-glycine sequences and, in this sequence context, asparagine is known to be particularly susceptible to deamidation. Two-dimensional gel electrophoresis revealed a complex series of VP6 isoforms with an apparent molecular mass of approximately 45,000 Da and a pI ranging from 5.25 to 5.8. This pattern could partly be explained by the potential for deamidation at several sites within the protein.

Original languageEnglish
Pages (from-to)12-24
Number of pages13
JournalFunctional and Integrative Genomics
Volume1
Issue number1
DOIs
Publication statusPublished - 2000

Keywords

  • acetylation
  • deamidation
  • modifications
  • peptide mass fingerprinting
  • rotavirus
  • serotype classification

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