Automated 20 kpsi RPLC-MS and MS/MS with Chromatographic Peak Capacities of 1000-1500 and Capabilities in Proteomics and Metabolomics

摘要

Proteomics analysis based-on reversed-phase liquid chromatography (RPLC) is widely practiced; however,variations providing onning-edge RPLC performance have generally not been adopt6d even though their benefits arewell established. Here, we describe an automated format 20 kpsi RPLC system for proteomics and metabolomics that includes on-line coupling of micro-solid phase extraction for sample loading and allows electrospray ionizationemitters to be readily replaced. The system uses 50 mum i.d. x 40-2O0 cm fused-silica capillaries packed with1.4-3-mum porous C18-bonded silica particles to obtain chromatographic peak capacities of 1000-1500 forcomplex peptide and metabolite mixtureses. This separation quality provided high-confidence identifications of > 12 000 different tryptic peptides from >2000 distinct shewanella oneidensis proteins (approx40percent of the proteins predicted for the S. oneidensis proteome) in a single 12-h ion trap tandem mass spectrometry (MS/MS) analysis. The protein identification reproducibility approached 90percent between replicate experiments. The average protein MS/MS identification rate exceeded 10 proteins/min, and 1207 proteins were identified in 12O min through assignmentof 5944 different peptides. The proteomic analysis dynamic range of the 20 kpsi RPLC-ion trap MS/MS was approximately 10~6 based on analyses of a human blood plasma sample, for which 835 distinct proteins were identified with high confidence in a single 12-h run. A single run of the 20 kpsi RPLC-accurate mass MS detected > 5000 different compounds from a metabolomics sample.