Mechanistic Aspects of On‐Line Electrochemical Tagging of Free L ‐Cysteine Residues during Electrospray Ionisation for Mass Spectrometry in Protein Analysis

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.