On-line coupling of micro-enzyme reactor with micro-membrane chromatography for protein digestion, peptide separation, and protein identification using electrospray ionization mass spectrometry

To miniaturize high-performance membrane chromatography, a poly(vinylidene fluoride) membrane medium, employed as the stationary phase, is sandwiched between two poly (dimethylsiloxane) substrates containing the microchannels. The microchannels are fabricated by the capillary molding technique, involving the use of capillaries as the channel template and the fluid inlet/outlet. The micro(mu)-membrane chromatography system is coupled with a mu -enzyme reactor containing immobilized trypsins for performing rapid protein digestion. peptide separation, and protein identification using electrospray ionization mass spectrometry. Separation performance of cytochrome c digest in mu -membrane chromatography is compared with the results obtained from a regular reversed-phase mu -liquid chromatography. The efficacy and the potentials of R-membrane chromatography in tryptic mapping are reported. On-line integration of the mu -enzyme reactor with mu -chromatographic separation techniques and electrospray ionization mass spectrometry clearly provides a microanalytical platform for automated sample handling, minimized sample loss, and reduced sample consumption. It also provides enhanced detection sensitivity and dynamic range for the analysis of complex protein mixtures such as cell lysates in proteornics research. (C) 2001 Elsevier Science B.V. All rights reserved.