No abstract

We present the state-of-the-art in miniaturized sample preparation, immunoassays, one-dimensional and multidimensional analyte separations, and coupling of microdevices with electrospray ionization-mass spectrometry. Hyphenation of these different techniques and their relevance to proteomics will be discussed. In particular, we will show that analytical performances of microfluidic analytical systems are already close to fulfill the requirements for proteomics, and that miniaturization results at the same time in a dramatic increase in analysis throughput. Throughout this review, some examples of analytical operations that cannot be achieved without microfluidics will be emphasized. Finally, conditions for the spreading of microanalytical systems in routine proteomic labs will be discussed.

A microfabrication process leading to a sheathless electrospray interface for mass spectrometry analysis is described. Photoablation is performed on a polymer substrate, allowing the integration of a thick-film conductive track in a sealed microchannel. High voltage is supplied close to the outlet, through an embedded microelectrode. The microspray is generated directly from the edge of the substrate without any tip addition. The flexibility of this technology provides a wide range of dimensions for the probe and the microelectrode design, including location, shape, and conductive material used. Thanks to the thick-film microelectrode and the hydrophobicity of the polymer, which avoids solution spreading at the outlet, the device has been found to be an efficient ionization source providing a stable MS signal through time. Moreover, the same device can be used several times without failure. The performance of the microspray has been studied in simple infusion mode for proteins and reserpine MS analyses. The detection limit of reserpine was found to be at the picomolar level in full-scan MS mode. It implies also that approximately 500 zmol was read consumed during 3 min of infusion. A dynamic range from pico- to millimolar level is also underlined.

No abstract

This paper demonstrates the coupling of a plasma etched polymer microfluidic system with an electrospray mass spectrometer by generation of a nanospray. Taking advantage of the microtechnology processes and polymer properties, high volume production with good reproducibility of hydrophobic interfaces could be obtained. The nanospray was directly produced from the outlet of the plastic microfabricated chip positioned in front of the capillary entrance of the mass spectrometer. No chemical background due to the polymer has been observed under standard nanospray conditions. The performances of the spray as well as its efficiency have been demonstrated by flow measurements, stability establishment and tandem mass spectrometry experiment on angiotensin II. The spray was actuated without additional flow in methanol: water:acetic acid (50:49:1%) solution. A 40 fmol/microL detection limit could be reached.

An overview of the electrospray processes is given and discussed, from the formation and the onset of the spray to the generation of gas-phase ions. This soft ionisation method for mass spectrometry is indeed characterized by non-equilibrium conditions, and the striking features relative to its use are difficult to be analytically or numerically modelled or even observed. In particular, the ionisation model known as the ''ion evaporation model'' will be largely discussed and its relevance with respect to the other model for gas-phase ions generation, the ''charge residue model'' will be highlighted. Lastly, the influence of the species nature and respective concentrations in the sprayed solution will be reviewed.

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.

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. (J Am Soc Mass Spectrom 2004, 15, 1767-1779