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Mechanistic Aspects of On‐Line Electrochemical Tagging of Free L ‐Cysteine Residues during Electrospray Ionisation for Mass Spectrometry in Protein Analysis
Author(s) -
Roussel Christophe,
Rohner Tatiana C.,
Jensen Henrik,
Girault Hubert H.
Publication year - 2003
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.200390031
Subject(s) - chemistry , cysteine , protonation , mass spectrometry , adduct , benzoquinone , hydroquinone , electrospray , electrospray ionization , cyclic voltammetry , electrochemistry , stereochemistry , chromatography , organic chemistry , enzyme , ion , electrode
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.