On-line protein digestion and peptide mapping by capillary electrophoresis with post-column labeling for laser-induced fluorescence detection.
A nanoliter enzyme microreactor was developed for on-line capillary electrophoresis (CE) peptide mapping of proteins, allowing picomole quantities of proteins to be digested. The enzyme microreactor was formed by immobilizing trypsin onto a monolithic capillary column, which was prepared by in situ polymerization of glycidyl methacrylate and ethylene dimethacrylate in a capillary. Highly efficient digestion of three protein standards was demonstrated. The detection of peptide fragments in CE was enhanced by post-column derivatization and laser-induced fluorescence detection. The microreactor has a volume of about 30 nL and is coupled with a separation capillary via a fluid joint for on-line digestion. The overall analysis, including digestion and separation, lasted only about 16 min. Column efficiencies > 300 000 plates/m were obtained for most peaks in the electropherogram of an on-line peptide mapping experiment of denatured alpha-lactalbumin under optimal conditions.
Ye
Mingliang
M
Department of Chemistry, University of Washington, Seattle, WA, USA.
Hu
Shen
S
Schoenherr
Regine M
RM
Dovichi
Norman J
NJ
D000818:Animals; D019075:Electrophoresis, Capillary; D004852:Epoxy Compounds / Q000737:chemistry; D005453:Fluorescence; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry*; D007834:Lasers; D008689:Methacrylates / Q000737:chemistry; D010449:Peptide Mapping; D014357:Trypsin / Q000737:chemistry*
D004852:Epoxy Compounds; D008689:Methacrylates; C004919:ethylene dimethacrylate; D007768:Lactalbumin; D014357:Trypsin; C007870:glycidyl methacrylate