Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics.
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.
Shen
Y
Y
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Zhao
R
R
Belov
M E
ME
Conrads
T P
TP
Anderson
G A
GA
Tang
K
K
Pasa-Tolić
L
L
Veenstra
T D
TD
Lipton
M S
MS
Udseth
H R
HR
Smith
R D
RD
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
D020543:Proteome; D010450:Endopeptidases