Of gels, offgel and off with the gel

In the context of prefractionation methods for proteomics, this work deals mainly with the development of electrophoretic tools for isoelectric focusing of peptides and proteins for the analysis of biological mixtures. In the light of existing devices for isoelectric focusing (IEF), the objective has been to develop multicompartment devices, designed for the IEF of peptides and proteins. The first choice of the OFFGEL for IEF among other techniques is justified by the easy recovery of liquid fractions of peptides, of small volumes, further amenable to liquid chromatography (as a second dimension separation) or mass spectrometry analyses. The resolution is a key point to consider in the design of separation units. Finite element simulation of the isoelectric focusing of peptides by OFFGEL has allowed the design of a multicompartment OFFGEL device for high resolution separation of peptides. The numerical simulations have highlighted the importance of the mobility near pI for the IEF kinetics and the final peak shape. The calculation of the distribution of peptides mobility near pI for three proteomes has allowed concluding on the optimal width of the well to obtain best separation. This mathematical study has also illustrated the high focusing power of the OFFGEL technique as a separation tool for shotgun proteomics application. The design of a multicompartment OFFGEL device was then done, based on the results of the simulations. The reproducibility of the pH gradient was validated, the loading capacity was evaluated for proteins, and a demonstration of the high resolution separation of peptides and proteins from a complex biological mixture was performed. The OFFGEL separation was then integrated in a workflow combining chemical tagging of the cysteine residues. This approach showed that the high resolution of peptide OFFGEL and the added information on the sequence of the peptides permitted a more confident and accurate protein identification. In the context of gel-free proteomics, another separation cell has been designed, that enables performing isoelectric focusing without the need of an immobilized pH gradient (IPG) gel. The novel device has been characterized in terms of performances and has been be applied to a biological sample of Escherichia coli, showing a more rapid separation of proteins than OFFGEL IEF, thus demonstrating its potential for fast proteome prefractionation purposes. An electrochemical cell has also been designed, for the transfer of ionizable species by electrochemistry at the micro-interface of two immiscible electrolytes (µ-ITIES), supported by an IPG gel as the aqueous phase and a small drop of organic phase. This study was initially motivated by the aim of performing online extraction of proteins/peptides during IEF separation. The use of this device for the transfer of model molecules was demonstrated, opening the door to further developments concerning the electrochemical transfer of proteins.

Scanning Electrochemical Microscopy and Finite Element Modeling of Structural and Transport Properties of Electrochemical Systems

Dmitry Momotenko

Understanding the fundamental properties of electrochemical systems, especially at the microscopic level, requires advanced tools combining electrochemical microscopy and scanning probe techniques with mathematical modeling for the quantification of the physicochemical characteristics of these environments. Comparison, analysis and sometimes calibration of the experimental results with theoretically predicted values is an indispensable strategy of any scientific method. In this thesis, experimental investigations are complemented with computations and numerical simulations. Novel microfluidic probes for scanning electrochemical microscopy (SECM) were developed. These soft SECM probes are equipped with an ultramicroelectode and integrated counter-reference electrode for characterizing the electrochemical reactivity of the substrate. They also incorporate microfluidic channels for delivery and removal of electrolyte solutions to and away from the probe tip allowing SECM experiments to be carried out on initially dry substrates. Additional integration of this concept with a chemical detection of the circulating electrolyte by means of mass-spectrometric analysis demonstrated the possibility to turn these microfluidic probes into a scanning tool for localized stimulation, control and read-out of chemical events occurring at interfaces. This work was also accomplished with two- and three- dimensional finite element modeling of the amperometric probe response under various geometrical arrangements taking into account microfluidic perturbations. Theoretical investigations of the capacitive properties of liquid/liquid interfaces were carried out taking into account the effect of smooth variation of permittivity across the liquid/liquid boundary. The width and symmetry of the resulting interphase were shown to be crucial characteristics for the variation of ion solvation within the interfacial region and fitting the experimental data with a finite element model was used to estimate the thickness of liquid/liquid junctions. Diode-like behavior of tapered charged nanopores was investigated by means of computing ion fluxes using a simple perm-selective approximation and more complicated finite element models taking into account a uniform surface charge density inside the conical pipettes. These simulations provided an insight on the mechanisms governing the rectifying behavior of such systems under various circumstances, i.e. geometrical arrangement, electrolyte concentrations and different electrical potential/voltage scan rate conditions, proving the existence of a perm-selective barrier attributed to diffuse layer overlapping inside the charged nanopore. Finally, theoretical investigations of the analytical characteristics of solid phase extraction-gradient elution-mass spectrometry (SPE-GEMS) technique for proteomics were performed. The computations were used to optimize the experimental conditions and to estimate performance aspects of this microchip-based strategy by following sorption-desorption equilibria under gradient elution conditions, i.e. by varying solvent strength.

EPFL2013

Photoproduction of Hydrogen by Decamethylruthenocene

Lucie Josette Renée Rivier

Splitting water to produce hydrogen (H2) fuel appears very promising to address the challenges of solar energy storage and global warming as the only by-product generated is oxygen (O2). An alternative strategy, called batch water-splitting, based on Interfaces between Two Immiscible Electrolyte Solutions (ITIES) and artificial light-induced water-splitting is presented in the following thesis. This approach consists of two biphasic systems for the photo-production of H2 and O2. The following thesis focuses on the realisation of the H2 side. In first instance, the photo-induced hydrogen evolution reaction (HER) by decamethylruthenocene (Cp2Ru(II)) is reported as a strategy to facilitate water splitting in biphasic systems. Hydrogen evolution by Cp2Ru(II) was studied in detail. The study highlights that Cp2Ru(II) is an attractive molecule capable of photo-reducing hydrogen without the need for an additional sensitizer. Electrochemical, gas chromatographic and spectroscopic (UV/vis, 1H and 13C NMR) measurements indicate that the production of hydrogen occurs by a two-step process. First, the decamethylruthenocene hydride ([Cp2Ru(IV)(H)]+) is formed in the presence of acids, followed by the reduction of this complex via a hetero-dissociation reaction leading to a first release of hydrogen. Thereafter, the resultant decamethylruthenocenium ion ([Cp2Ru(III)]+) is further reduced leading to a second release of hydrogen by subtraction of a proton from a methyl group of [Cp2Ru(III)]+. Experimental results showed an excitation of [Cp2Ru(IV)(H)]+ at ï¬ = 243 nm to evolve H2 for the first oxidation. [Cp2Ru(III)]+ was produced from the reduction of protons by Cp2Ru(II) at ï¬ = 365 nm and electrochemically regenerated in situ on a Fluorinated Tin Oxide (FTO) electrode surface. A promising internal quantum yield of 25 % was obtained for HER by Cp2Ru(II) combined with electrochemical recycling. Thereafter, HER by Cp2Ru(II) was performed at ITIES. Shake-flask experiments demonstrated the production of H2 only when the biphasic system was positively polarized, to favor proton transfer. Kinetics/thermodynamics for decamethylruthenocene hydride formation were electrochemically evaluated at liquidÇliquid interface. Simulated curves developed using COMSOL Multiphysics software and compared to experimental data, indicate a modified EC (electrochemicalâchemical) mechanism for the [Cp2Ru(IV)(H)]+ formation at polarised interfaces. In the proposed pathway, [Cp2Ru(IV)(H)]+ is sufficiently stable in dichloroethane to transfer at negative potentials to the aqueous phase where it quickly dissociates. Additionally, the SHG response of [Cp2Ru(IV)(H)]+ as function of the polarisation applied confirmed this mechanism. Finally, an alternative method using homogeneous catalysts, Co(dmgh)2(py)Cl and Fe2(ï-SCH2C6H4CH2S)(CO)6 at liquidÇliquid interfaces was investigated. Coupled with a sacrificial electron donor and a sensitizer, H2 production was achieved for both catalysts. However, results showed the polarisation was not responsible for the proton transfer and the electron donor was identified as the dominant proton source. This study represents major progress in the development of the batch water splitting process as it overcomes the use of sacrificial electron donors. Moreover, these investigations provide substantial improvement in the general understanding of the photo-production of H2 by metallocenes and reactions and characterisations at ITIES.

EPFL2017

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.

EPFL2006