Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification

An integrated platform is presented for rapid and sensitive protein identif ication by on-line protein digestion and analysis of digested proteins usin g electrospray ionization mass spectrometry or transient capillary isotacho phoresis/capillary zone electrophoresis with mass spectrometry detection. A miniaturized membrane reactor is constructed by fabricating the microfluid ic channels on a poly(dimethylsiloxane) substrate and coupling the microflu idics to a poly(vinylidene fluoride) porous membrane with the adsorbed tryp sin. On the basis of he large surface area-to-volume ratio of porous membra ne media, adsorbed trypsin onto the poly(vinylidene fluoride) membrane is e mployed for achieving ultrahigh catalytic turnover. The extent of protein d igestion in a miniaturized membrane reactor tan be directly controlled by t he residence time of protein analytes inside the trypsin-adsorbed membrane, the reaction temperature, and the protein concentration. The resulting pep tide mixtures can either be directly analyzed using electrospray ionization mass spectrometry or further concentrated and resolved by electrophoretic separations prior to the mass spectrometric analysis, This microfluidic sys tem enables rapid identification of proteins in minutes instead of hours, c onsumes very little sample (nanogram or less), and provides on-line interfa ce with upstream protein separation schemes for the analysis of complex pro tein mixtures such as cell lysates.