Preparation and characterization of a packed bead immobilized trypsin reactor integrated into a PDMS microfluidic chip for rapid protein digestion

This paper demonstrates the design, efficiency and applicability of a simple, inexpensive and high sample throughput microchip immobilized enzymatic reactor (IMER) for rapid protein digestion. The IMER contains conventional silica particles with covalently immobilized trypsin packed inside of a poly(dimethylsiloxane) (PDMS) microchip channel (10 mm x 1 mm x 35 mu m). The microchip consists of 9 different channels, enabling 9 simultaneous protein digestions. Trypsin was covalently immobilized using carbodiimide activation, the ideal trypsin/silica particle ratio (i.e. measured mass ratio before the immobilization reaction) was determined. The amount of immobilized trypsin was 10-15 mu g trypsin for 1 mg silica particle. Migration times of CZE peptide maps showed good repeatability and reproducibility (RSD%=0.02-0.31%). The IMER maintained its activity for 2 months, in this period it was used effectively for rapid proteolysis. Four proteins (myoglobin, lysozyme, hemoglobin and albumin) in a wide size range (15-70 kDa) were digested to demonstrate the applicability of the reactor. Their CZE peptide maps were compared to peptide maps obtained from standard in-solution digestion of the four proteins. The number of peptide peaks correlated well with the theoretically expected peptide number in both cases, the peak patterns of the electropherograms were similar, however, digestion with the microchip IMER requires only < 10 s, while in-solution digestion takes 16 h. LC-MS/MS peptide mapping was also carried out, the four proteins were identified with satisfying sequence coverages (29-50%), trypsin autolysis peptides were not detected. The protein content of human serum was digested with the IMER and with in-solution digestion.