Off-line coupling of preparative capillary zone electrophoresis with microwave-assisted acid hydrolysis and matrix-assisted laser desorption ionization mass spectrometry for protein sequencing.
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).
Department of Chemistry, Gunning/Lemieux Chemistry Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
Mahmoud M
MM
Yassine
Nan
N
Guo
Hongying
H
Zhong
Liang
L
Li
Charles A
CA
Lucy
D000143:Acids; D045304:Cytochromes c / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D006868:Hydrolysis; D008872:Microwaves; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000143:Acids; D045304:Cytochromes c