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

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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.