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Abstract

Modification strategies targeting specific amino acids in proteins are widespread in proteomic analysis. Cysteine residues have received deep consideration in view of their nucleophilic properties and their occurrence in the proteome. A recently developed micro-electrospray emitter for mass spectrometry was used to electrogenerate species reactive towards specific residues in biomolecules. When spraying L-cysteine in the presence of hydroquinone, the thiol cysteine moiety reacts via a 1,4-Michael addition with the benzoquinone electrochemically generated at the electrode. A series of electrogenerated selective electrophiles based on substituted benzoquinones was characterized as tags for L-cysteine. The rate constants pertaining to the addition of L-cysteine onto the benzoquinones were determined through electrochemical techniques. It was shown that the rate constants are primarily dependent on the electronic nature of the substituents. The apparent tagging extents observed for L-cysteine in microspray mass spectrometry experiments were shown to be highly dependent of the ionization efficiencies of the tag. The on-line mass spectrometric electrochemical tagging (EC-tagging) of cysteine residues was studied for peptides. The EC-tagging was tested with the different hydroquinones on an undecapeptide containing one cysteine residue. Methoxycarbonyl-1,4-hydroquinone was shown to be the most efficient probe and revealed to be suitable to count cysteine units in peptides containing up to three cysteines. The number of cysteines corresponds to the number of characteristic mass shifts observed from the unmodified peptide. The identification of bovine serum albumin and human a-lactalbumin digest samples in a peptide mapping strategy were greatly improved by the application of the EC-tagging technique as post-column treatment. Indeed, the determination of cysteine content in the tryptic peptides provides powerful supplementary information to the masses. The tagging method was applied to the determination of four proteins in a model mixture. In parallel, the microspray emitter was characterized as an electrolysis flow cell for the EC-tagging of peptides. The Levich equation was validated as a first approximation for the calculation of the convection-diffusion limiting current in the device. A finite element simulation of the multi-tagging process of peptides was developed to yield the relative distribution and concentration of tags, untagged and tagged species in the microchannel. The main chemical parameters determining the kinetics of the labelling were assessed and discussed considering the microfluidic aspects of the process. The control of the tagging extent allows the simultaneous mass spectrometric analysis of both the unmodified and of the modified peptide(s). This theoretical work has established the range of optimum conditions for the determination of the number of cysteines in peptides containing up to five cysteine groups. The mass spectrometric EC-tagging of cysteine residues in proteins was studied to probe the cysteine environment. An analytical model was developed to calculate rapidly the tagging extent before the spray event. Experiments with unmodified proteins and their chemically reduced forms have highlighted the strong effect of the cysteine site reactivity on the tagging efficiencies. This study has shown relevant parameters for such on-line electrochemical derivatization / mass spectrometric detection strategies. The chemical derivatization of cysteines by benzoquinone reagents was also investigated. These alkylating reagents revealed efficient for diagonal liquid chromatography to isolate cysteinyl peptides by the retention time shifts due to the hydrophobicity of the tags. The work has demonstrated that the inherent electrochemistry of the electrospray can be employed as post column treatment to derivatize cysteinyl biomolecules. Analytical strategies have been developed to take advantage of this electrochemically-controlled modification.

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