title: Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics.
abstract: 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-micron) C18 bonded particles provided a total peak capacity of approximately 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 micrograms) could be loaded onto packed capillaries of 150-micron 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.
authors.affiliation: Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Shen
authors.forename: R
authors.initials: R
authors.lastname: Zhao
authors.forename: M E
authors.initials: ME
authors.lastname: Belov
authors.forename: T P
authors.initials: TP
authors.lastname: Conrads
authors.forename: G A
authors.initials: GA
authors.lastname: Anderson
authors.forename: K
authors.initials: K
authors.lastname: Tang
authors.forename: L
authors.initials: L
authors.lastname: Pasa-Tolić
authors.forename: T D
authors.initials: TD
authors.lastname: Veenstra
authors.forename: M S
authors.initials: MS
authors.lastname: Lipton
authors.forename: H R
authors.initials: HR
authors.lastname: Udseth
authors.forename: R D
authors.initials: RD
authors.lastname: Smith
mesh_terms: D002853:Chromatography, Liquid; D017357:Cyclotrons; D010450:Endopeptidases; D005051:Eubacterium / Q000737:chemistry / Q000166:cytology; D005583:Fourier Analysis; D006868:Hydrolysis; D020543:Proteome / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D020543:Proteome; D010450:Endopeptidases