Automated 20 kpsi RPLC-MS and MS/MS with Chromatographic Peak Capacities of 1000-1500 and Capabilities in Proteomics and Metabolomics
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- 作者：Yufeng Shen ; Rui Zhang ; Ronald J. Moore ; Jeongkwon Kim ; Thomas O. Metz ; Kim K. Hixson ; Rui Zhao ; Eric A. Livesay ; Harold R. Udseth ; and Richard D. Smith
- 刊名：Analytical Chemistry
- 出版年：2005
- 出版时间：May 15, 2005
- 年：2005
- 卷：77
- 期：10
- 页码：3090 - 3100
- 全文大小：780K
- 年卷期：v.77,no.10(May 15, 2005)
- ISSN：1520-6882
文摘
Proteomics analysis based-on reversed-phase liquid chromatography (RPLC) is widely practiced; however, variations providing cutting-edge RPLC performance havegenerally not been adopted even though their benefits arewell established. Here, we describe an automated format20 kpsi RPLC system for proteomics and metabolomicsthat includes on-line coupling of micro-solid phase extraction for sample loading and allows electrospray ionizationemitters to be readily replaced. The system uses 50 
mi.d. × 40-200 cm fused-silica capillaries packed with1.4-3-m porous C18-bonded silica particles to obtainchromatographic peak capacities of 1000-1500 forcomplex peptide and metabolite mixtures. This separationquality provided high-confidence identifications of >12 000different tryptic peptides from >2000 distinct Shewanella oneidensis proteins (~40% of the proteins predictedfor the S. oneidensis proteome) in a single 12-h ion traptandem mass spectrometry (MS/MS) analysis. The proteinidentification reproducibility approached 90% betweenreplicate experiments. The average protein MS/MS identification rate exceeded 10 proteins/min, and 1207proteins were identified in 120 min through assignmentof 5944 different peptides. The proteomic analysis dynamic range of the 20 kpsi RPLC-ion trap MS/MS wasapproximately 106 based on analyses of a human bloodplasma sample, for which 835 distinct proteins wereidentified with high confidence in a single 12-h run. Asingle run of the 20 kpsi RPLC-accurate mass MS detected >5000 different compounds from a metabolomicssample.
mi.d. × 40-200 cm fused-silica capillaries packed with1.4-3-m porous C18-bonded silica particles to obtainchromatographic peak capacities of 1000-1500 forcomplex peptide and metabolite mixtures. This separationquality provided high-confidence identifications of >12 000different tryptic peptides from >2000 distinct Shewanella oneidensis proteins (~40% of the proteins predictedfor the S. oneidensis proteome) in a single 12-h ion traptandem mass spectrometry (MS/MS) analysis. The proteinidentification reproducibility approached 90% betweenreplicate experiments. The average protein MS/MS identification rate exceeded 10 proteins/min, and 1207proteins were identified in 120 min through assignmentof 5944 different peptides. The proteomic analysis dynamic range of the 20 kpsi RPLC-ion trap MS/MS wasapproximately 106 based on analyses of a human bloodplasma sample, for which 835 distinct proteins wereidentified with high confidence in a single 12-h run. Asingle run of the 20 kpsi RPLC-accurate mass MS detected >5000 different compounds from a metabolomicssample.