Reproducibility of a solid-phase trypsin microreactor for peptide mapping by capillary electrophoresis

We describe the rapid, facile fabrication of an immobilized protease microreactor and its characterization for peptide mapping by capillary electrophoresis (CE). A 30 cm long fused silica capillary was dry-packed with trypsin-immobilized controlled pore glass (CPG) beads. A 40 mu l solution of protein standard (beta-casein or insulin chain B) was perfused through the microreactor for ca. 2 h by applying low pressure at the inlet. The digest was collected at the microreactor outlet and the tryptic fragments were separated by CE and detected by UV absorbance using a diode array detector. Tryptic peptide maps obtained by solid-phase digestion in the microreactor were compared to those obtained by traditional, liquid-phase methods to evaluate the microreactor's performance and extent of trypsin autoproteolysis (self-digestion). The effect of substrate flow rate through the packed bed of trypsin-CPG (i.e. the residence time) and effect of substrate concentration were also investigated. In general, digestion conditions in the microreactor were very reproducible, whereas CE separation conditions were found to contribute the most to migration time variability in peptide mapping. Reproducible peptide maps were obtained for 40 Cll aliquots of a 2 mg ml(-1) beta-casein solution. The activity of the immobilized enzyme was high enough to ensure re-usability of this microreactor for at least 10 digestions. (C)2000 Elsevier Science B.V. All rights reserved.