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

Abstract

Proteomics is the large-scale study of the proteins related to a genome. Presently, 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 expensive and time-consuming and, for this reason, have been typically paid insufficient attention. However, optimization becomes a critical issue as we try to analyze ever shrinking amounts of proteins. We present here the development of a technique that allows the rapid, sensitive, semiquantitative, and automated optimization of the processes involved in proteomics. Furthermore, it allows the rapid testing of new methodologies without having to rely on expensive mass spectrometric techniques. The technique, based on head column stacking capillary zone electrophoresis, allows the concentration, separation, and analysis of protein digests at concentrations from high picomoles to subfemtomoles per microliter and sample volumes from a few microliters to a few hundred microliters produced by proteomic processes. Furthermore, the incorporation of UV detection in the system allows the tracking of the relative changes in peptide levels observed during optimization. In addition, all the buffers and solvents used in this technique are compatible with its future coupling to electrospray ionization mass spectrometry. The potential of this technique for the analysis of low-abundance proteins is demonstrated using peptide standards and tryptic digests of standard proteins. Moreover, we exemplify the application of this technique in proteomic prototyping for the rapid and automated study of the procedure of enzymatic digestion of proteins.