High-Efficiency Capillary Isoelectric Focusing of Peptides

Several approaches are presently being developed for global proteome characterization that are based upon the analysis of polypeptide mixtures resulting from digestion of (often complex) mixtures of proteins. Improved methods for peptide analysis are needed that provide for sample concentration, higher resolution separations, and direct compatibility with mass spectrometry. In this work, methods for the high-efficiency capillary isoelectric focusing (CIEF) separation of peptides have been developed that provide for simultaneous sample concentration and separation according to peptide isoelectric point. Under typical nondenaturing CIEF conditions, peptides are concentrated ∼500-fold, and peptides present at <1 ng/μL were detectable using conventonal UV detection. CIEF separations of peptides provided much faster measurements of isoelectric points compared with conventional isoelectric focusing in gels. Very small differences in peptide isoelectric points (ΔpI ∼0.01) could be resolved. High-efficiency CIEF separations for complex peptide mixtures from tryptic digestion of yeast cytosol fractions were obtained and showed significant improvement over those obtained using capillary zone electrophoresis and packed capillary reversed-phase liquid chromatography.