Techniques for the optimization of proteomic strategies based on head column stacking capillary electrophoresis

Proteomics is the large-scale study of the proteins related to a genome. Pr esently, proteomic procedures have relied on mass spectrometry as a tool of choice to perform analysis of proteins. Optimization and understanding of the different steps involved in proteomics using mass spectrometry is expen sive and time-consuming and, for this reason, have been typically paid insu fficient attention. However, optimization becomes a critical issue as we tr y to analyze ever shrinking amounts of proteins. We present here the develo pment of a technique that allows the rapid, sensitive, semiquantitative, an d automated optimization of the processes involved in proteomics. Furthermo re, it allows the rapid testing of new methodologies without having to rely on expensive mass spectrometric techniques. The technique, based on head c olumn stacking capillary zone electrophoresis, allows the concentration, se paration, and analysis of protein digests at concentrations from high picom oles to subfemtomoles per microliter and sample volumes from a few microlit ers to a few hundred microliters produced by proteomic processes. Furthermo re, the incorporation of UV detection in the system allows the tracking of the relative changes in peptide levels observed during optimization. In add ition, all the buffers and solvents used in this technique are compatible w ith its future coupling to electrospray ionization mass spectrometry. The p otential of this technique for the analysis of low-abundance proteins is de monstrated using peptide standards and tryptic digests of standard proteins . Moreover, we exemplify the application of this technique in proteomic pro totyping for the rapid and automated study of the procedure of enzymatic di gestion of proteins.