Off-line coupling of preparative capillary zone electrophoresis with microwave-assisted acid hydrolysis and matrix-assisted laser desorption ionization mass spectrometry for protein sequencing

Abstract

An off-line coupling of capillary electrophoresis (CE) with microwave-assisted acid hydrolysis/matrix-assisted laser desorption ionization mass spectrometry (MAAH/MALDI) has been developed for protein identification and characterization. Preparative scale protein separations enable collection of 10–50 pmol of purified cytochrome c for subsequent sequencing using MAAH/MALDI. To reduce protein adsorption onto the silica surface, the cationic surfactant-based coatings, dimethylditetradecylammonium bromide and dimethyldioctadecylammonium bromide, are employed. The choice of the buffer conditions is critical for both the preparative CE and MAAH/MALDI method. The use of high buffer concentrations (100 mM Bis–tris) reduces electromigration dispersion, but suppressed MALDI ionization such that a peptide sequence coverage of only 80% was achieved at a sample loading of 40 g L −1 of each cytochrome c. By reducing the buffer concentration to 25 mM Bis–tris, the sequence coverage increased to 95% at a sample loading of 40 g L −1.