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(μ)-membrane chromatography system is coupled with a μ-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 μ-membrane chromatography is compared with the results obtained from a regular reversed-phase μ-liquid chromatography. The efficacy and the potentials of μ-membrane chromatography in tryptic mapping are reported. On-line integration of the μ-enzyme reactor with μ-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 proteomics research.