Development of Electrokinetically Based Two-Dimensional Separation Methodologies for Proteomic Studies

Proteomic studies require analytical methods capable of separating, identifying and quantifying thousands of proteins. Consequently, developing separation methodologies with high resolving power necessary to separate complex protein samples prior to protein identification via mass spectrometry (MS) has become a major challenge in proteomic studies. It is now demonstrated that a single separation technique is unable to provide a peak capacity sufficient to resolve complex protein mixtures, therefore multidimensional separation methodologies have to be developed. In this context, the highest peak capacity is obtained when the hyphenated methods present separation mechanisms as different as possible. Subsequently, the orthogonality of a two-dimensional (2-D) separation methodology has to be also evaluated as a feature that expresses the efficiency of hyphenation. The objectives of the thesis were mainly focused on the development and application of 2-D separation methodologies in proteomic studies. In the first part of this work, the potential of the combination of Capillary zone electrophoresis (CZE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is assessed. To further provide a bioanalytical platform with high-sensitivity capabilities, field-enhanced sample injection is integrated as on online preconcentration strategy upstream from the electrokinetic separation. Then, a novel approach to assess the orthogonality of 2-D separation systems based on conditional entropy is introduced. It considers the quantitative distribution of peaks in the entire separation space such that the orthogonality obtained is independent of the number of peaks observed for each separation technique. As another part of this work, the hyphenation of off-gel electrophoresis (OGE) with CZE is revised. While the application of the developed 2-D separation methodology is expanded to protein scale, a significant improvement of the practical peak capacity for peptide separation is also obtained. In this chapter, a computer based model is also developed which is able to digest a specific protein and simulate the 2-D OGE-CZE separation map of resulting peptides. Afterwards, a practical protocol for proteolysis assisted by trypsin loaded NH2-MOSF during OGE separation is developed. Besides savings in experimental effort and enhanced proteolysis efficiency, the straightforward assessment of pI values of peptides after simultaneous digestion and separation with OGE which facilitates peptide identification, is also one valuable aspect of the developed methodology. In the last part of the thesis, the proof of concept of the application of a push-pull microfluidic device for electrotransfer of proteins from a separation gel to a membrane is presented. The membrane can be then subjected to proteolysis and MALDI-MS analysis to visualize with high resolution the protein sample separated.

Orthogonality of Two-Dimensional Separations Based on Conditional Entropy

Hubert Girault, Mohammad Karzand, Mohammad Reza Pourhaghighi

A new approach to assess the orthogonality of two-dimensional (2-D) separation systems based on conditional entropy is developed. It considers the quantitative distribution of peaks in the entire separation space such that the orthogonality obtained is independent of the number of peaks observed for each separation technique. Therefore, it can be used to compare the orthogonality of different 2-D separation protocols for a given sample. Herein, the developed method has been employed to estimate the orthogonality of peptide separation by off-gel electrophoresis (OGE) hyphenated to capillary zone electrophoresis (CZE).

American Chemical Society2011

Miniaturized analytical systems for mass spectrometry-based protein studies

Mélanie Abonnenc

Current proteomic strategies depend strongly on the development of analytical methodologies and instrumentation. In parallel to the development of mass spectrometry (MS) - based proteomic workflows, microfluidic devices emerged in this field as a flexible tool for rapid and sensitive protein studies. In this context, the present work focuses on the development of miniaturized analytical systems for protein studies, especially by electrospray ionization mass spectrometric detection. Several approaches have been proposed to complement the mass spectrometric analysis of tryptic peptides using chemical tagging to isolate specific subclasses of proteins by affinity baits or for quantitation purposes. Optimization of a chemical tagging reaction in a sandwich mixer-reactor that consists of mixing two solutions providing the reactants in a channel, one central laminar flow being sandwiched between two outer flow solutions, was first investigated by finite-element method. The numerical simulations have highlighted the importance of positioning the reactant presenting the lowest diffusion coefficient close to the sidewall, as well as decreasing the outer flow velocities, to enhance a chemical reaction because of the parabolic flow profile across the microchannel. This optimization is even more relevant for consecutive reactions. As infusion-based electrospray microchips are known to present great advantages in terms of sensitivity compared to standard ionization source, an ESI emitter microchip based on polymer photo-ablation was designed. First, the hyphenation of the chip in a LC-MS workflow was successfully achieved and evaluated by comparison with a standard pneumatically-assisted ESI source. To perform enhanced on-chip post-column derivatization of peptides, a substitute design to the sandwich mixer-reactor was explored to improve reaction in a microchip before ESI – MS analysis. The electrospray micromixer chip includes a passive mixing unit to perturbate the flow along the microchannel. The mixing efficiency was demonstrated by fluorescence imaging, and on-chip chemical derivatization of peptides and kinetic studies before mass spectrometry were achieved. The on-line LC-MS derivatization of cysteinyl peptides was shown to provide a more confident and accurate protein identification by adding information on the peptidic sequence. Within the development of electrospray microchips for mass spectrometry, many efforts have been put forward in the hyphenation of functional units. Immobilization of functionalized stationary phase in microfluidic systems is widely used to achieve protein or peptide isolation, sample cleaning, separation or reaction. In this context, magnetic beads have proven to be quite useful compared to other methods, such as immobilized packed beads or monoliths, because of their high and reversible magnetization, and various surface functionalization. A polymer microchip including a magnetic track array was designed to focus the magnetic field generated by permanent magnets towards precise locations along the microchannel. A multi-plug magnetic bead capture was obtained and a significant increase of bead capture efficiency was demonstrated both numerically and experimentally, which is beneficial for affinity separation applications, as example. The research in microfluidic front-end devices for mass spectrometry is actually oriented to the development of multi-spray interfaces for high-throughput analysis. An electrospray microchip with a multi-track array was developed. The capability of the device to screen alternatively up to six samples and to perform relative quantification was assessed. Soft ionization techniques have revolutionized the analysis of proteins by mass spectrometry. In contrast with the major content of the thesis dealing with microfluidic systems for protein analysis, the last chapter presents a novel ambient ionization method, so-called membrane-desorption electrospray ionization (M-DESI). The principle is similar to the desorption electrospray ionization (DESI) method. But, in the M-DESI approach the sample is desorbed directly from a mesh membrane positioned vertically and in-axis between the ESI emitter and the mass spectrometer inlet. Analysis of peptides and proteins was demonstrated as a proof-of-concept, offering great perspectives for detection after separation on membrane.

EPFL2009

Thiol-targeted microspray mass spectrometry of peptides and proteins through on-line EC-tagging

Loïc Dayon

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.