High-Throughput Proteomics Using High-Efficiency Multiple-Capillary Liquid Chromatography with On-Line High-Performance ESI FTICR Mass Spectrometry Yufeng Shen, Nikola Tolic ´, Rui Zhao, Ljiljana Pas ˇa-Tolic ´, Lingjun Li, Scott J. Berger, Richard Harkewicz, Gordon A. Anderson, Mikhail E. Belov, and Richard D. Smith* Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352 We report on the design and application of a high- efficiency multiple-capillary liquid chromatography (LC) system for high-throughput proteome analysis. The mul- tiple-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 arrange- ment 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 electro- spray ionization (ESI) sources were operated independ- ently and can be used either for “backup” operation or for parallel operation with other mass spectrometers. This high-efficiency multiple-capillary LC system utilizes switch- ing valves for all operations, enabling automated opera- tion. The separation efficiency of the dual-capillary column arrangement, optimal capillary dimensions (column length and packed particle size), capillary regeneration condi- tions, and mobile-phase compositions and their compat- ibility with electrospray ionization were investigated. A high magnetic field (1 1 .4 T) Fourier transform ion cyclo- tron 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 ∼6 5 0 , and the 2 -D capillary LC-FTICR analysis provided a combined resolving power of >6 × 10 7 components. For yeast cytosolic tryptic digests >100 000 polypeptides were detected, and ∼1000 pro- teins could be characterized from a single capillary LC- FTICR analysis using the high mass measurement accu- racy (∼1 ppm) of FTICR, and likely more if LC retention time information were also exploited for peptide identi- fication. The “postgenomic era” presents the challenge of analyzing the complex array of proteins (i.e., the proteome) expressed by an organism, tissue, or cell to aid in the understanding of the operation of complex cellular pathways, networks, and “modules” under various physiological conditions. 1-5 An organism’s proteome is not fixed, but changes with the state of the development, the tissue, and the environmental conditions. 6,7 To delineate key proteins and unravel the complex molecular pathways and networks involved in cellular responses, a set of proteomes in response to various environmental “perturbation” can be analyzed and exploited. This requires that the proteome analysis methodol- ogy be sensitive, robust, quantitative, and high-throughput. Mass spectrometry (MS) is playing an increasingly important role in proteome analysis, 8-10 and current proteome analysis strategies primarily involve its combination with two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) separations of proteins. 11-13 However, limitations of 2-D PAGE that arise due to measurement dynamic range, protein solubility issues, and extremes of protein isoelectric points and molecular weights have impeded complete proteome characterization, 6,14 and thus true proteome-wide analysis will only be realized by implementation of more effective approaches that likely will include higher resolution and/ or multidimensional separation strategies. In one approach for improving proteome coverage, Yates and co-workers demonstrated the use of global protein digests with two-dimensional capillary liquid chromatography (LC/ LC) coupled (1) Wilkins, M. R.; Williams, K. L.; Appel, R. D.; Hochstrasser, D. F. Proteome Research: New Frontiers in Functional Genomics; Springer: Berlin, Germany, 1997. (2) Uddhav, K.; Ketan, S. Mol. Biol. Rep. 1998 , 25, 27-43. (3) Celegans Sequencing Consortium, Genome Sequence of the Nematode C. elegans: A Platform for Investigating Biology, Science 1998 , 282, 2012- 2018. (4) Adams, M. D. Bioassays 1996 , 18, 261-262. (5) Anderson, L.; Seilhammer, J. Electrophoresis 1997 , 18, 533-537. (6) Harry, J.; Wilkins, M. R.; Herbert, B. R.; Packer, N. H.; Gooley, A. A.; Williams, K. L. Electrophoresis 2000 , 21, 1071-1081. (7) Anderson, L.; Seihammer, J. Electrophoresis 1997 , 18, 533-537. (8) Klose, J. Electrophoresis 1999 , 20, 643-652. (9) Yates, J. R. J. Mass Spectrom. 1998 , 33,1-19. (10) Gevaert, K.; Vandekerckhove, J. Electrophoresis 2000 , 21, 1145-1154. (11) Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe, C. Proc. Natl. Acad. Sci. U.S.A. 1993 , 30, 5011-5015. (12) Brown, R. S.; Lennon, J. J. Anal. Chem. 1995 , 67, 1998-2003. (13) Yates, J. R.; McCormack, A. L.; Eng. J. Anal. Chem. 1996 , 68, 534-540. (14) Gygi. S. P.; Corthals, G. L. Zhang, Y. Rochon, Y.; Aebersold, R. Proc. Natl. Acad. Sci. U.S.A. 2000 , 97, 9390-9395. Anal. Chem. 2001, 73, 3011-3021 10.1021/ac001393n CCC: $20.00 © 2001 American Chemical Society Analytical Chemistry, Vol. 73, No. 13, July 1, 2001 3011 Published on Web 05/16/2001