Reactivity of the NS2/3(907-1206)ASK4 protein with β-mercaptoethanol studied by electrospray ion trap mass spectrometry

Laura Orsatti, Michele Pallaoro, Christian Steinküler, Stefania Orru', Fabio Bonelli

Research output: Contribution to journalArticle

Abstract

The present work reports a mass spectrometric investigation of the NS2/3 protein, a protease from hepatitis C virus (HCV). During routine protein manipulation, in the presence of 100 mM β-mercaptoethanol and under denatured conditions, the protein was unexpectedly modified at its cysteine residues, and the increased molecular weight corresponded to one molecule of β- mercaptoethanol bound. The modified protein, once refolded, was found to be less active than the unmodified one. The aim of this work was to investigate whether the reactivity of cysteines with β-mercaptoethanol involves one specific, highly reactive residue of the sequence, or if the modification is a random process. Liquid chromatography (LC) coupled on-line with an electrospray ion trap mass spectrometer was used to identify the modification sites. It was found that five cysteines out of nine had reacted with β-mercaptoethanol, none of them showing a significantly higher reactivity than the others. 95% of sequence coverage was obtained.

Original languageEnglish
Pages (from-to)1919-1927
Number of pages9
JournalRapid Communications in Mass Spectrometry
Volume16
Issue number20
DOIs
Publication statusPublished - 2002

ASJC Scopus subject areas

  • Analytical Chemistry
  • Spectroscopy

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