In proteomic analysis of complex samples at the peptide level (termed shotgun proteomics), an effective prefractionation is crucial to decrease the complexity of the peptide mixture for further analysis. In this perspective, the high-resolving power of the IEF fractionation step is a determining parameter, in order to obtain well-defined fractions and correct information on peptide isoelectric points, to provide an additional filter for protein identification. Here, we explore the resolving power of OFFGEL IEF as a prefractionation tool to separate peptides. By modeling the peak width evolution versus the peptide charge gradient at pI, we demonstrate that for the three proteomes considered in silico (Deinococcus radiodurans, Saccharomyces cerevisiae, and Homo sapiens), 90% of the peptides should be correctly focused and recovered in two wells at most. This result strongly suggests OFFGEL to be used as a powerful fractionation tool in shotgun proteomics. The influence of the height and shape of the compartments is also investigated, to give the optimal cell dimensions for an enhanced peptide recovery and fast focusing time.

An experimental setup to study the pH dependence of standard ion-transfer potentials at the water/NPOE interface is presented. The system is composed of a microhole generated by laser photoablation in a 12-µm polyethylene terephthalate film, the aqueous phase consisting of a commercial immobilized pH gradient gel reswelled in electrolyte solution and a droplet of organic solution. Two electrodes are used, an Ag/AgCl aqueous electrode and an Ag/AgTPBCl organic electrode. This setup is applied to the study of two ionizable compounds (pyridine, 2,4-dinitrophenol). Thermodynamic parameters such as the standard transfer potential, the Gibbs energy of transfer, and the partition coefficients of the ionized forms as well as the neutral forms of these drug compounds are evaluated by differential pulse voltammetry. The data obtained are summarized in ionic partition diagrams, which are a useful tool for predicting and interpreting the transfer behavior of ionized drugs at the liquid/liquid interfaces mimicking the biological membranes.Recently, the electrochemical study of the transfer of ionic species across the interface between two immiscible electrolyte solutions (ITIES) has gained great significance due to its wide applicability in different fields such as ion-selective electrodes for application to amperometric sensors, solvent extraction, drug lipophilicity, and its consequences on drug delivery. 1-4 Unlike numerous traditional chromatographic and potentiometric systems developed to study the distribution of ionic species and giving an indirect access to partition coefficients, voltammetry at the ITIES allowed us to evaluate the standard partition coefficient of both the neutral and the ionized forms. 5,6 The four-electrode system initially introduced by Samec et al. 7 was later used intensively by Reymond et al. 2 to study the transfer of many ionizable drugs at the interface between water and 1,2-dichloroethane (DCE), and the introduction of ionic partition diagrams revealed a most interesting aspect for the study of drug lipophilicity. 8 Indeed, a partition diagram of a specific drug between two immiscible liquids is a representation of the conditions corresponding to the predominance of different forms of the compound (basic, neutral, acid) as a function of the Galvani potential difference and the pH of the aqueous phase. This representation has been revealed to be a useful tool to mimic the passage of a drug through a biological membrane and help understand the action of that drug.However, the initial systems used to study the distribution of ionic species between two immiscible solutions used large ITIES, requiring quite large volumes of each phase 2 . Thus, when only limited amount of species is available, micro-ITIES are more suitable, such as liquid/liquid (L/L) interfaces supported at the tip of micropipets 9,10 or systems using a droplet of organic phase or aqueous phase. 11,12 For example, Gobry et al. 13 reported experiments with an aqueous droplet supported at an Ag/AgCl disk electrode covered wi...

A minidevice for performing gel‐free proteome prefractionation via conventional IEF in soluble carrier ampholyte buffers is reported here. It consists of a compact block of polyoxymethylene in which eight samples and two electrode chambers are machined. Each of the eight sample chambers can be filled with up to 120 μL of sample and has the following size: 7 mm width, 3 mm depth and 10 mm height. The anodic and cathodic compartments have the same width and height as the sample chambers, but with a depth of 6 mm, thus accepting up to 250 μL of electrodic solutions. Focusing is in general accomplished in 2 h with a voltage gradient of up to 1000 V (7 cm electrode distance). Easy fractionation and collection of the content of the eight chambers is achieved by simply pressing a rubber diaphragm against the edges of the thin walls separating each well, this automatically breaking liquid continuity. The performance of this device has been tested by subfractionating total cell lysates of a human cancer cell line (U2Os) and of Escherichia coli bacterial cells, and by analysing the content of each chamber by mono‐dimensional SDS‐PAGE and 2‐D maps.

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