Integration of immobilized trypsin bead beds for protein digestion within a microfluidic chip incorporating capillary electrophoresis separations andan electrospray mass spectrometry interface

A microfluidic device is described in which an electrospray interface to a mass spectrometer is integrated with a capillary electrophoresis channel, a n injector and a protein digestion bed on a monolithic substrate. A large c hannel, 800 mu m wide, 150 mu m deep and 15 mm long, was created to act as a reactor bed for trypsin immobilized on 40-60 mu m diameter beads. Separat ion was performed in channels etched 10 mu m deep, 30 mu m wide and about 4 5 mm long, feeding into a capillary, attached to the chip with a low dead v olume coupling, that was 30 mm in length, with a 50 mu m i.d. and 180 mu m o.d. Sample was pumped through the reactor bed at flow rates between 0.5 an d 60 mu L/min. The application of this device for rapid digestion, separati on and identification of proteins is demonstrated for melittin, cytochrome c and bovine serum albumin (BSA), The rate and efficiency of digestion was related to the flow rate of the substrate solution through the reactor bed. A flow rate of 1 or 0.5 mu L/min was found adequate for complete consumpti on of cytochrome c or BSA, corresponding to a digestion time of 3-6 min at room temperature. Coverage of the amino acid sequence ranged from 92% for c ytochrome c to 71% for BSA, with some missed cleavages observed. Melittin w as consumed within 5 s, In contrast, a similar extent of digestion of melit tin in a cuvet took 10-15 min. The kinetic limitations associated with the rapid digestion of low picomole levels of substrate were minimized using an integrated digestion bed with hydrodynamic flow to provide an increased ra tio of trypsin to sample. This chip design thus provides a convenient platf orm for automated sample processing in proteomics applications. Copyright ( C) 2000 John Wiley & Sons, Ltd.