Packed Capillary Reversed-Phase Liquid Chromatography with High-Performance Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Proteomics

• Published in: Analytical chemistry (Washington) 2001-04, Vol.73 (8), p.1766-1775
• Main Authors: Shen, Yufeng, Zhao, Rui, Belov, Mikhail E, Conrads, Thomas P, Anderson, Gordon A, Tang, Keqi, Paša-Tolić, Ljiljana, Veenstra, Timothy D, Lipton, Mary S, Udseth, Harold R, Smith, Richard D
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Liquid; Cyclotrons; Endopeptidases; Eubacterium - chemistry; Eubacterium - cytology; Fourier Analysis; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Hydrolysis; Proteins; Proteome - analysis; Spectrometry, Mass, Electrospray Ionization
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac0011336

In this study, high-efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-μm) C18 bonded particles provided a total peak capacity of ∼1000 for cellular proteolytic polypeptides when interfaced with an ESI-FTICR mass spectrometer under composition gradient conditions at a pressure of 10 000 psi. Large quantities of cellular proteolytic digests (e.g., 500 μg) could be loaded onto packed capillaries of 150-μm inner diameter without a significant loss of separation efficiency. Precolumns with suitable inner diameters were found useful for improving the elution reproducibility without a significant loss of separation quality. Porous particle packed capillaries were found to provide better results than those containing nonporous particles because of their higher sample capacity. Two-dimensional analyses from the combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of >1 million polypeptide components and simultaneously provided information for the identification of the separated components based upon the accurate mass tag concept previously described.