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

Abstract

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 approximately 650, and the 2-D capillary LC-FTICR analysis provided a combined resolving power of > 6 x 10(7) components. For yeast cytosolic tryptic digests > 100,000 polypeptides were detected, and approximately 1,000 proteins could be characterized from a single capillary LC-FTICR analysis using the high mass measurement accuracy (approximately 1 ppm) of FTICR, and likely more if LC retention time information were also exploited for peptide identification.

An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics

“…The system was designed and tested for operation up to 10,000 psi. The basic system is an optimized, automated, and a more robust advanced version of a previously described system [26]. The goals for the LC and fluidics system were to simultaneously provide the following: (1) Very high performance, (2) very high pressure, (3) high levels of robustness and reliability, and (4) capabilities for high throughput and continuous, 24/7 operations.…”

“…The ChemInert units valves (Valco) were critical for both chromatographic performance and system reliability in continuous, 24/7 operations. They are significantly improved versions of the positive feedback ChemInert valves, first described by Shen et al [26] for use in high-efficiency LC-FTICR MS analyses of proteomics samples, incorporating an advanced polymer material in the rotor seal. This new material permits high-pressure operations (10,000 or 20,000 psi) and improved stability of the internal flow channel in the rotor seal, resulting in increased injection cycles and longer service life at high pressures.…”

“…The mobile phase is a water-acetonitrile gradient. Columns were packed as described by Shen et al [26]. Stationary phase specifications for the analytical column were as follows.…”

“…When coupled to capillary LC separation [20], FTICR mass spectrometry [21,22] has been shown to be an ultra-sensitive approach with high dynamic range, mass resolution, and mass accuracy for characterization of proteolytic digests [23][24][25][26]. The sensitivity provided by FTICR has been shown to be increased by ion trapping and accumulation in a 2D rf-only octopole trap positioned external to an FTICR mass spectrometer [27].…”

Algorithms for automatic interpretation of high resolution mass spectra

“…Due to the high mass accuracy and resolving power of an FTMS, MALDI-FTMS [5][6][7] and ESI-FTMS [8,9] are becoming the instruments of choice for proteomics [10], and experiments on proteins and large fragments of proteins, so called "top-down" [11][12][13][14] mass spectrometry. These experiments tend to slow down due to the lack of sophisticated methods for automatic spectrum analysis.…”