Integrated Platform for Proteome Profiling with Combination of Microreversed Phase Based Protein and Peptide Separation via Online Solvent Exchange and Protein Digestion

An online integrated platform for proteome profiling was established, with the combination of protein separation by microreversed phase liquid chromatography (μRPLC), online acetonitrile (ACN) removal, and pH adjustment by a hollow fiber membrane interface (HFMI), online digestion by an immobilized enzymatic microreactor (IMER), as well as peptide separation and proteins identification by μRPLC or nano-RPLC-electrospray ionization tandem mass spectrometry (μRPLC-ESI-MS/MS). To evaluate the performance of such a platform, a three-protein mixture with mass ranging from 5 to 500 ng was analyzed automatically. Compared to the offline counterpart, although similar protein sequence coverages were obtained by the integrated platform, the signal intensity of total ion chromatogram was improved by almost 4 times. In addition, such an integrated platform was further applied for the analysis of extracted proteins from rat brain. Compared to the results obtained by offline counterpart and traditional MudPIT approach under similar conditions, by the integrated platform, the identified protein group number was comparable, but the analysis time was shortened to less than half of that taken by the traditional approaches. All these results demonstrated that our developed integrated platform might offer a promising tool for high-throughput and large-scale profiling of proteomes.