High-Throughput Proteomics Using High-Efficiency Multiple-Capillary Liquid Chromatography with On-Line High-Performance ESI FTICR Mass Spectrometry

We report on the design and application of a high-efficiency multiple-capillary liquid chromatography (LC) system for high-throughput proteome analysis. The multiple-capillary LC system using commercial LC pumps was operated at a pressure of 10 000 psi to deliver mobile phases through a novel passive feedback valve arrangement that permitted mobile-phase flow path switching and efficient sample introduction. The multiple-capillary LC system uses several serially connected dual-capillary column devices. The dual-capillary column approach eliminates the time delays for column regeneration (or equilibration) since one capillary column was used for a separation while the other was being washed. Several serially connected dual-capillary columns and electrospray ionization (ESI) sources were operated independently and can be used either for “backup” operation or for parallel operation with other mass spectrometers. This high-efficiency multiple-capillary LC system utilizes switching valves for all operations, enabling automated operation. The separation efficiency of the dual-capillary column arrangement, optimal capillary dimensions (column length and packed particle size), capillary regeneration conditions, and mobile-phase compositions and their compatibility with electrospray ionization were investigated. A high magnetic field (11.4 T) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was coupled on-line with this high-efficiency multiple-capillary LC system using an ESI interface. The capillary LC provided a peak capacity of ∼650, and the 2-D capillary LC−FTICR analysis provided a combined resolving power of >6 × 107 components. For yeast cytosolic tryptic digests >100 000 polypeptides were detected, and ∼1000 proteins could be characterized from a single capillary LC-FTICR analysis using the high mass measurement accuracy (∼1 ppm) of FTICR, and likely more if LC retention time information were also exploited for peptide identification.