Mechanistic aspects of on-line electrochemical tagging of free L-cysteine residues during electrospray ionisation for mass spectrometry in protein analysis

Chemphyschem. 2003 Feb 17;4(2):200-6. doi: 10.1002/cphc.200390031.

Abstract

The mechanistic details behind an electrochemically induced tagging of L-cysteine residues in peptides and proteins have been unravelled using cyclic voltammetry. It was found that when hydroquinone is oxidised in the medium used in electrospray ionisation mass spectrometry (ESI-MS) a protonated form of benzoquinone is produced that acts as an efficient electrophile for free L-cysteine residues. Upon substitution of L-cysteine the reduced form of the adduct is formed, which may be further oxidised leading to further substitution of L-cysteine. Digital simulations of the cyclic voltammograms corroborated the mechanism and allowed a determination of the homogeneous second order rate constant corresponding to the addition of L-cysteine onto the protonated form of benzoquinone. The selectivity of the tagging process was confirmed using ESI-MS, which showed that a protein without L-cysteine residues does not react with benzoquinone dissolved in the medium. Finally, the kinetic information obtained in this investigation is used to discuss the optimal parameters for a nanospray capable of quantitative tagging of L-cysteine residues.

MeSH terms

  • Animals
  • Benzoquinones / chemistry
  • Cysteine / chemistry*
  • Electrochemistry / methods
  • Humans
  • Hydroquinones / chemistry
  • Kinetics
  • Molecular Probes / chemistry*
  • Online Systems
  • Oxidation-Reduction
  • Proteins / analysis*
  • Spectrometry, Mass, Electrospray Ionization / methods*

Substances

  • Benzoquinones
  • Hydroquinones
  • Molecular Probes
  • Proteins
  • quinone
  • Cysteine
  • hydroquinone