Probing Cysteine Reactivity in Proteins by Mass Spectrometric EC-Tagging (original) (raw)
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Physical Chemistry Chemical Physics, 2005
On-line electrogeneration of mass tags in a microspray emitter is used to quantify the number of cysteine groups in a given peptide. A finite-element simulation of the multi-step process yields the relative distribution and concentration of tags, untagged and tagged species in the microchannel before the spray event. The work focuses on the tagging of cysteine moieties in peptides or proteins by electrogenerated quinone mass probes. The main chemical parameters determining the kinetics of the labelling are assessed and discussed considering the microfluidic aspects of the process. The control of the tagging extent allows the simultaneous MS analysis of both the unmodified and modified peptide(s). The number of cysteine groups corresponds to the number of characteristic mass shifts observed from the unmodified peptide. The present theoretical work establishes the range of optimum conditions for the determination of the number of cysteine groups in peptides containing up to five cysteine groups.
Electrophoresis, 2005
The electrochemically induced mass spectrometric tagging of cysteines by substituted hydroquinones was studied for peptides in a classical electrospray solvent (i.e., MeOH/H2O/AcOH 50/49/1). The tagging efficiency was tested with different hydroquinone compounds on an undecapeptide containing one cysteine residue. 2-Carboxymethylhydroquinone was the most reactive probe and revealed to be suitable for cysteine quantification in peptides containing up to three cysteine residues, even in the case of two consecutive cysteines in the sequence. We demonstrate the compatibility of the on-line electrochemical tagging method for the cysteine content analysis of peptides coming from gel-free protein digestion procedures. The identification of bovine serum albumin and human α-lactalbumin digest samples in a peptide mapping strategy was greatly improved by the application of the electrotagging technique as post-column treatment. Indeed, the determination of cysteine content in the tryptic peptides provided powerful information in order to enhance the identification score as well as the discrimination against other protein candidates. The tagging method was then applied to the determination of four proteins in a model mixture.
Journal of The American Society for Mass Spectrometry, 2004
We present herein a review of our work on the on-line electrochemical generation of mass tags toward cysteine residues in peptides and proteins. Taking advantage of the inherent electrochemical nature of electrospray generated from a microfabricated microspray emitter, selective probes for cysteine were developed and tested for on-line nonquantitative mass tagging of peptides and proteins. The nonquantitative aspect of the covalent tagging thus allows direct counting of free cysteines in the mass spectrum of a biomolecule through additional adduct peaks. Several substituted hydroquinones were investigated in terms of electrochemical properties, and their usefulness for on-line mass tagging during microspray experiments were assessed with L-cysteine, peptides, and intact proteins. Complementarily, numerical simulations were performed to properly understand the respective roles of mass transport, kinetics of electrochemical-chemical reactions, and design of the microspray emitter in the mass tagging overall efficiency. Finally, the on-line electrochemical tagging of cysteine residues was applied to the analysis of tryptic peptides of purified model proteins for protein identification through peptide mass fingerprinting.
CHIMIA International Journal for Chemistry, 2004
The electrochemically induced mass spectrometric tagging of cysteines by substituted hydroquinones was studied for peptides in a classical electrospray solvent (i.e., MeOH/H 2 O/AcOH 50/49/1). The tagging efficiency was tested with different hydroquinone compounds on an undecapeptide containing one cysteine residue. 2-Carboxymethylhydroquinone was the most reactive probe and revealed to be suitable for cysteine quantification in peptides containing up to three cysteine residues, even in the case of two consecutive cysteines in the sequence. We demonstrate the compatibility of the on-line electrochemical tagging method for the cysteine content analysis of peptides coming from gel-free protein digestion procedures. The identification of bovine serum albumin and human a-lactalbumin digest samples in a peptide mapping strategy was greatly improved by the application of the electrotagging technique as post-column treatment. Indeed, the determination of cysteine content in the tryptic peptides provided powerful information in order to enhance the identification score as well as the discrimination against other protein candidates. The tagging method was then applied to the determination of four proteins in a model mixture.
ChemPhysChem, 2003
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.
Journal of Electroanalytical Chemistry, 2004
A series of electrogenerated selective electrophiles based on substituted benzoquinones has been characterized as tags for l-cysteine and cysteine residues in proteins. The electrophiles are generated electrochemically from the corresponding hydroquinones. It is shown from mass spectrometry analysis that the electrogenerated benzoquinone can tag the biomolecules. The rate constants pertaining to the addition of l-cysteine onto the electrogenerated benzoquinones have been determined using electrochemical techniques. The substitution patterns have been unraveled leading to the assessment of site-specific rate constants. It is shown that the rate constants are primarily dependent on the electronic nature of the substituents as expressed by the Hammett substitution constant. The apparent tagging yields observed for l-cysteine in nanospray mass spectrometry experiments do not correspond to the yields expected from the electrochemical study, as the ionisation efficiencies are highly dependent on the tag. Finally, the on-line tagging has been tested using β-lactoglobulin A and myoglobin. Based on these results, it is concluded that the tagging reaction is selective towards cysteine when it takes place in the nanospray interface. The results show that the methodology presented can be used for a rapid characterization and identification of reactive sites in biomolecules.
On-line electrochemical tagging of cysteines in proteins during nanospray
Electrochemistry Communications, 2002
An electrochemical modification of free cysteine residues is studied and characterized by means of quinone addition. Taking advantage of the electrolytic nature of electrospray interfaces (ESI), an electrochemical tagging is performed prior to mass spectrometry (MS) analyses. The tagging has been studied by MS and different mechanisms, involving electrochemical and/or chemical steps, could be characterized. It is demonstrated that the present nanospray is a very efficient tool to obtain cysteine modification. Using the high voltage electrode of the nanospray interface to perform protein specific tagging is a novel method that can be associated to analytical or preparative techniques, such as digestion of proteins or capillary electrophoresis, for post-column modifications.
Thiol-targeted microspray mass spectrometry of peptides and proteins through on-line EC-tagging
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-cy...
Journal of Mass Spectrometry, 2006
Multitrack electrospray chips (MTEC) were fabricated by UV-photoablation of polyethylene terephthalate (PET) substrates. They are composed of an array of up to six microchannels that are successively used as electrospray ionization (ESI) emitters for mass spectrometry (MS). There is no requirement for alignment of the different spraying microchannels with the mass spectrometer orifice. The MTEC is thus fixed in front of the mass spectrometer and the successive MS analyses are performed without moving the chip. The sequential electrospraying by successive application of an identical high voltage in each off-axis microchannel was evaluated for the fast screening of peptides and proteins. The counting of cysteines in peptides through chemical modification and the relative quantification of a peptide in two samples are presented herein as two original strategies based on this new analytical tool. Copyright © 2006 John Wiley & Sons, Ltd.
Molecules
High complexity of cell and tissue proteomes limits the investigation of proteomic biomarkers. Therefore, the methods of enrichment of some chemical groups of peptides including thiopeptides are important tools that may facilitate the proteomic analysis by reducing sample complexity and increasing proteome coverage. Here, we present a new method of cysteine-containing tryptic peptide enrichment using commercially available TentaGel R RAM resin modified by the linker containing the maleimide group, allowing thiol conjugation. The captured tryptic peptides containing lysine residue were then tagged by 2,4,6-triphenylpyrylium salt to form 2,4,6-triphenylpyridinium derivatives, which increases the ionization efficiency during mass spectrometry analysis. This makes it possible to conduct an ultrasensitive analysis of the trace amount of compounds. The proposed strategy was successfully applied in the enrichment of model tryptic podocin peptide and podocin tryptic digest.