Post-column fluorescence derivatization of proteins and peptides in capillary electrophoresis with a sheath flow reactor and 488 nm argon ion laser excitation

Abstract

We report the use of a sheath flow reactor for post-column fluorescence derivatization of proteins. The derivatization reaction employed naphthalene-2,3-dicarboxaldehyde (NDA) and β-mercaptoethanol, which were added in the sheath buffer. The labeled proteins were detected by laser-induced fluorescence with an argon-ion laser beam at 488 nm. The performance of this detection scheme was evaluated by separation of some protein standards. A column efficiency of 450,000 plates/m was obtained without stacking. The limits of detection for those standard proteins were determined to be from 8 to 32 nM. Excellent linear relationship was obtained with correlation coefficient of 0.9998 for α-lactalbumin concentration ranging from 3.91×10 −7 to 1.25×10 −5 M. Separation of protein standards at low pH was also demonstrated by reversing the electroosmotic flow (EOF) with addition of cetyltrimethylammonium bromide (CTAB) to the running buffer. Different separation selectivity was achieved, but the sensitivity is poorer than that at high pH. This post-column derivatization detection system was applied successfully to analyze the protein extract from HT29 human colon cancer cells as well as tryptic peptides.