On‐line protein digestion and peptide mapping by capillary electrophoresis with post‐column labeling for laser‐induced fluorescence detection

Abstract

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.

Vinyl functionalized silica hybrid monolith-based trypsin microreactor for on line digestion and separation via thiol-ene “click” strategy

“…Covalent immobilization of enzyme on a capillary monolithic column is an useful method to prepare enzyme microreactor, e.g., immobilization by genetic engineering [24], Staudinger ligation reaction [25], or azide-alkyne click reaction [26]. The immobilized trypsin microreactor by coupling with RPLC-MS/MS, CE and CE-MS has been applied for analysis of proteins and peptides [27][28][29][30]. Thiol-ene or thiol-acrylate polymerization was used in biological researches [1,4,5,7].…”

Recent advances in capillary separations for proteomics

“…Ye and co-workers [40] have constructed a liquid junction for on-line combination of a separation capillary with a nanoliter-sized trypsin reactor formed by immobilizing trypsin within a monolithic capillary column. In contrast to the studies by Kato et al [39], the liquid joint contained within a Lexan and parafilm device allowed the use of different buffers for the optimization of protein digestion and peptide separation steps.…”

LIF detection of peptides and proteins in CE

“…This postcolumn labelling technique was satisfactorily applied to a protein extract from HT29 human colon cancer cells and tryptic peptides using a pH of 10 for the separation. It was also used to detect peptide fragments resulting from trypsic protein digestion in a nanolitre immobilized trypsin reactor coupled with the separation capillary [106].…”