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 chrom atography (RPLC) coupled on-line with high-performance Fourier transform io n cyclotron resonance (FTICR) mass spectrometry has been investigated for t he characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-mum) C18 bonded particles provided a total peak capacity of similar to 1000 for cellular proteolytic polypeptide s when interfaced with an ESI-FTICR mass spectrometer under composition gra dient conditions at a pressure of 10 000 psi, Large quantities of cellular proteolytic digests (e.g,, 500 mug) could be loaded onto packed capillaries of 150-mum inner diameter without a significant loss of separation efficie ncy. Precolumns with suitable inner diameters were found useful for improvi ng the elution reproducibility without a significant loss of separation qua lity. Porous particle packed capillaries were found to provide better resul ts than those containing nonporous particles because of their higher sample capacity. Two-dimensional analyses from the combination of packed capillar y RPLC with high-resolution FTICR yield a combined capacity for separations of >1 million polypeptide components and simultaneously provided informati on for the identification of the separated components based upon the accura te mass tag concept previously described.