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.

Generation of mass tags by the inherent electrochemistry of electrospray for protein mass spectrometry

“…To obtain more accurate values of the different kinetic constants k, the evolutions of the I p /I p 0 ratios versus the CVs scan rate were fitted to the experimental data, as shown in Figure 5b for hydroquinone 1c. The value of k was found to increase from 1a to 1d (see Table 1) [66,68]. This result could easily be explained by the fact that the coupling reaction involves a nucleophile (L-cysteine 3a=): in that case, the presence of an electron donor group such as CH 3 or CH 3 O increases the electronic density on the benzoquinone ring and is expected to decrease its reactivity toward L-cysteine addition.…”

“…) [66] was high enough to obtain a significant tagging yield. For this low value of chemical rate, the microchannel should be long enough to provide a suitable residence time (here t ϭ 2s) to let the species react before reaching the Taylor cone.…”

“…From an electrochemical point of view, the hydroquinone CVs are irreversible, exhibiting a slow electrode kinetic process. Moreover, CV of pure L-cysteine 3a= revealed the nonelectroactive character of this compound in the experimental conditions [66,68]. The electrochemical tagging of L-cysteine by hydroquinones 1a-d was then investigated by cyclic voltammetry in the MS spray medium: the CVs obtained with pure hydroquinones (oxidation current intensity I p 0 ) were compared with those of the same hydroquinones in the presence of L-cysteine (oxidation current intensity I p ).…”

“…Based on recent works on the electrochemical detection of sulfides [60,61], thiols [62], and cysteine [63,64] with quinone derivatives, we focused on the potential use of electrogenerated benzoquinones (coming from the oxidation of hydroquinones) able to react specifically with cysteine residues during electrospray mass spectrometric measurements. We present below a review of the different steps of the development of on-line electrochemical probes for cysteine mass tagging, from the proof of principle [65] to the elucidation of the tagging reaction mechanism [66], characterization of the microspray design influence on the tagging process [67], search for an optimal electrochemical probe [68], and final application to protein identification [69].…”

Electrochemical multi-tagging of cysteinyl peptides during microspray mass spectrometry: numerical simulation of consecutive reactions in a microchannel

“…The addition rate constants are assumed to be equal to those corresponding to the addition of L-cysteine on 1,4-benzoquinone and methoxycarbonyl-1,4-benzoquinone (210 and 5000 M À1 s À1 , respectively, in methanol-water-acetic acid 50 : 49 : 1). 26,27 For simplification, the diffusion coefficient of all the quinone probes is taken to be equal to 3.5 Â 10 À10 m 2 s À1 , which in fact corresponds to the diffusion coefficient of methoxycarbonyl-1,4-hydroquinone in methanol-water-acetic acid 50 : 49 : 1. 28 The mean diffusion coefficient of the target biomolecule P was chosen to be 1 Â 10 À10 m 2 s À1 .…”