Of gels, offgel and off with the gel: development of isoelectric focusing tools for proteomics

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.

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