Efficient capillary electrophoresis of peptides and proteins with bilayer-coated capillaries

Abstract

Peptides and proteins play a pivotal role in numerous life-processes and may show important pharmacological activities. There is an increasing interest in using these biomolecules for new therapies to treat life-threatening diseases. As a consequence, in the life sciences there is a great need for analytical techniques that allow reliable identification, characterization and quantification of peptides and proteins. Capillary electrophoresis (CE) is a powerful analytical technique that is well-suited for the analysis of biomolecules. CE offers much higher separation efficiencies than commonly used methods for peptides and proteins, such as HPLC and SDS-PAGE. Furthermore, it is relatively fast, and analyses can be performed under near-physiological and nondenaturing conditions. However, adsorption of peptides and proteins at the inner surface of fused-silica capillaries can result in poor separation efficiencies and irreproducible results. In the past, various methods have been proposed to circumvent this problem, but often these were laborious and showed limited applicability. Here we present a new elegant method that employs charged polymers to create a molecular bilayer on the capillary inner wall that prevents adsorption of biomolecular compounds. The coating procedure is straightforward and fast comprising only a simple flush of the capillary with aqueous solutions of the charged polymers. This thesis evaluates the suitability of capillaries coated with a bilayer of the polymers polybrene (PB) and poly(vinyl sulfonate) (PVS) for the analysis of peptides and proteins by CE with UV and MS detection. The electroosmotic flow induced by the coating was strong and constant, and independent of the type and pH of the BGE. The coating was stable providing reproducible migration-times for peptides and proteins (RSDs < 1.0%). The use of BGEs of high ionic strength effectively minimized interaction between analytes and the capillary wall, yielding plate numbers up to 600,000 for peptides and up to 300,000 for proteins. The bilayer coating demonstrated to be fully compatible with MS detection: no ion suppression or background signals of coating agents on mass spectra of the analytes were observed. Overall, these characteristics provide a CE system that is highly suitable for the fast, efficient and reproducible profiling of complex peptide and protein mixtures. The usefulness and resolving power of the new analytical system is demonstrated by the analyses of isomeric peptide/peptoid mixtures, closely-related peptides (enkephalins), and protein digests (revealing up to 20 peptides in a 1-min time interval). Furthermore, the bilayer-coated capillaries showed to be very useful for the stability monitoring and characterization of (degraded) biopharmaceuticals. These results indicate a performance that can be of great value in relevant fields such as proteomics, peptidomics, metabolomics, protein characterization, and quality control of protein pharmaceuticals preparations. In all these application areas, revealing differences among samples is an important issue, and requires the possibility to reliably acquire and compare separation profiles.