Nano-flow multidimensional liquid chromatography platform integrated with combination of protein and peptide separation for proteome analysis

An integrated multidimensional nano-flow liquid chromatography platform with the combination of protein and peptide separation via online digestion by an immobilized enzymatic reactor was established, and successfully applied for proteome analysis. By this platform, proteins were first separated by a weak anion and weak cation mixed-bed microcolumn under a series of salt steps, online digested by a trypsin immobilized enzymatic reactor, digests trapped and desalted by a C18 precolumn, separated by nano-reversed phase liquid chromatography, and finally identified by electrospray ionization-MS/MS. To evaluate the performance of such a platform, Escherichia coli whole cell lysate proteins were analyzed. Compared with the results obtained by shotgun approach, the identified protein number was increased by 6%, with the consumed time decreased from 38 to 14 h. We also compared with integrate platform based on micro-HPLC, and the required sample amount was decreased to 8 μg. These results demonstrated that such an integrated approach would be an attractive alternative to commonly applied approaches for proteome research.