High-Throughput Proteomics Using High Efficiency Multiple-Capillary Liquid Chromatography With On-Line High-Performance ESI FTICR Mass Spectrometry
- BATTELLE (PACIFIC NW LAB)
- ASSOC WESTERN UNIVERSITY
- Illinois Univ Of-Urbana/Champa
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 was operated at the pressure of 10,000 psi using commercial LC pumps to deliver the mobile phase and newly developed passive feedback valves to switch the mobile phase flow and introduce samples. The multiple-capillary LC system was composed of several serially connected dual-capillary column devices. The dual-capillary column approach was designed to eliminate the time delay for regeneration (or equilibrium) of the capillary column after its use under the mobile phase gradient condition (i.e. one capillary column was used in separation and the other was washed using mobile phase A). The serially connected dual-capillary columns and ESI sources were operated independently, and could be used for either''backup'' operation or with other mass spectrometer(s). This high-efficiency multiple-capillary LC system uses switching valves for all operations and is highly amenable to automation. The separations efficiency of dual-capillary column device, optimal capillary dimensions (column length and packed particle size), suitable mobile phases for electrospray, and the capillary re-generation were investigated. A high magnetic field (11.5 tesla) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was coupled on-line with this high-efficiency multiple-capillary LC system through an electrospray ionization source. The capillary LC provided a peak capacity of {approx}600, and the 2-D capillary LC-FTICR provided a combined resolving power of > 6 x 10 7 polypeptide isotopic distributions. For yeast cellular tryptic digests, > 100,000 polypeptides were typically detected, and {approx}1,000 proteins can be characterized in a single run.
- Research Organization:
- Pacific Northwest National Lab., Richland, WA (US)
- Sponsoring Organization:
- US Department of Energy (US)
- DOE Contract Number:
- AC06-76RL01830
- OSTI ID:
- 15001277
- Report Number(s):
- PNNL-SA-33921; KP1301030; TRN: US200401%%594
- Journal Information:
- Analytical Chemistry, Vol. 73, Issue 13; Other Information: PBD: 1 Dec 2000
- Country of Publication:
- United States
- Language:
- English
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