Integrated platform for proteome analysis with combination of protein and peptide separation via online digestion

An integrated platform with the combination of protein and peptide separation was established via online protein digestion, by which proteins were first separated by a microcolumn packed with mixed weak anion and weak cation exchange (WAX/WCX) particles under a series of salt steps, online digested by a trypsin immobilized microenzymatic reactor (IMER), trapped and desalted by two parallel C8 precolumns, separated by microreversed-phase liquid chromatography (μRPLC) under a linear gradient of organic modifier concentration, and finally identified by electrospray ionization-MS/MS (ESI-MS/MS). To evaluate the performance of such a platform, a mixture of myoglobin, cytochrome c, bovine serum albumin (BSA), and α-casein, with mass ranging from 25 ng to 2 μg, was analyzed. Compared to the methods by offline protein fractionation and shotgun based strategy, the analysis time, including sample preparation, digestion, desalting, separation, and detection, was shortened from ca. 30 to 5 h, and cytochrome c with abundance of 25 ng could be identified with improved sequence coverage. Furthermore, such an integrated platform was successfully applied into the analysis of proteins extracted from human lung cancer cells. Compared with the results obtained by the shotgun approach, the identified protein number was increased by 30%. All these results demonstrated that such an integrated approach would be an attractive alternative to commonly applied approaches for proteome research.