title: Mechanistic aspects of on-line electrochemical tagging of free L-cysteine residues during electrospray ionisation for mass spectrometry in protein analysis.
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.
authors.affiliation: Laboratoire d'Electrochimie Physique et Analytique Institut de Chimie Moléculaire et Biologique Ecole Polytechnique Fédérale de Lausanne 1015 Lausanne, Switzerland.
authors.forename: Christophe
authors.initials: C
authors.lastname: Roussel
authors.forename: Tatiana C
authors.initials: TC
authors.lastname: Rohner
authors.forename: Henrik
authors.initials: H
authors.lastname: Jensen
authors.forename: Hubert H
authors.initials: HH
authors.lastname: Girault
mesh_terms: D000818:Animals; D016227:Benzoquinones / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D004563:Electrochemistry / Q000379:methods; D006801:Humans; D006873:Hydroquinones / Q000737:chemistry; D007700:Kinetics; D015335:Molecular Probes / Q000737:chemistry*; D009862:Online Systems; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D016227:Benzoquinones; D006873:Hydroquinones; D015335:Molecular Probes; D011506:Proteins; C004532:quinone; D003545:Cysteine; C031927:hydroquinone