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Anal. Chem. 2001, 73, 3011-3021

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 Pasˇa-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 highefficiency 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 LCFTICR 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. The “postgenomic era” presents the challenge of analyzing the complex array of proteins (i.e., the proteome) expressed by an 10.1021/ac001393n CCC: $20.00 Published on Web 05/16/2001

© 2001 American Chemical Society

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 methodology 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, 20122018. (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.

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with tandem mass spectrometry (MS/MS).15,16 Approaches using global protease digestion have increased proteome coverage but have also increased the complexity of the mixture that must be addressed prior to and during MS analysis by as much as 100fold. The improved resolution obtained using multidimensional LC separations can more fully exploit the sensitivity achievable with conventional mass spectrometry (e.g., low-femtomole detection limits) and enabled the identification of many additional lower level proteins. The throughput of this type of approach, however, is significantly limited by the need for MS/MS analysis to identify each peptide, which ultimately constrains the total number of species that can be identified in a single LC-MS/MS analysis. Additionally, while the sensitivity of this approach represents an improvement over 2-D PAGE, there still exists a demand for greater sensitivity and dynamic range. To address these issues, we have developed a new strategy for global protein analysis that uses high-efficiency capillary LC combined with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry coupled through an electrospray ionization (ESI) interface, which permits proteomewide characterization and quantitation from a single experiment.17,18 The capillary LC-FTICR approach provides an opportunity to perform high-throughput proteome analyses but requires efficient (i.e., high duty cycle) LC operation.17 In this approach, peptides derived from global cellular proteolytic digests (e.g., using trypsin) are analyzed using a single dimension of highefficiency capillary LC coupled with FTICR, which offers unrivaled mass resolving power (m/∆m >105 for 5-10 kDa species), sensitivity (attomoles to zeptomoles), and mass measurements accuracy (MMA 106. The combined methodology holds the promise of proteome (15) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R. Nat. Biotechnol. 1999, 17, 676-682. (16) Washburn, M. P.; Wolters, D.; Yates, J. R. Nat. Biotechnol. 2001, 19, 242247. (17) Shen, Y.; Zhao, R.; Belov, M. E.; Conrads, T. P.; Anderson, G. A.; Tang, G.; Pasˇa-Tolic´, L.; Veenstra, T. D.; Lipton, M. S.; Udseth, H. R.; Smith, R. D. Anal. Chem. 2001, 73, 1766-1775. (18) Smith, R. D.; Pasˇa-Tolic´, L.; Lipton, M. S.; Jensen, P. K.; Anderson, G. A.; Shen, Y.; Conrads, T. P.; Udseth, H. R.; Harkewicz, R.; Belov, M. E.; Masselon, C.; Veenstra, T. D. Electrophoresis, in press. (19) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1-35. (20) Bruce, J. E.; Anderson, G. A.; Wen, J.; Harkewicz, R.; Smith, R. D. Anal. Chem. 1999, 71, 2595-2599. (21) Pasˇa-Tolic´, L.; Jensen, P. K.; Anderson, G. A.; Lipton, M. S.; Peden, K. K.; Martinovic´, S.; Tolic´, N.; Bruce, J. E.; Smith, R. D. J. Am. Chem. Soc. 1999, 121, 7949-7950. (22) Belov, M. E.; Gorshkov, M. V.; Udseth, H. R.; Anderson, G. A.; Smith, R. D. Anal. Chem. 2000, 72, 2271-2279. (23) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, SD-H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976. (24) Belov, M. E.; Nikolaev, E. N.; Anderson, G. A.; Auberry, A. J.; Harkewicz, R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2001, 12, 38-48.

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analyses with much greater throughput, sensitivity, dynamic range, and quantitative capability than presently achievable. In this work, we report methods for proteome analysis incorporating significantly improved high-efficiency capillary LC separations over those previously reported.17 The advances include the implementations of on-line sample introduction at 10 000 psi, a multiple-capillary column LC system to increase throughput and the capability for automation to allow efficient continuous operation. We illustrate the implementation of these modifications and further demonstrate the power of the combined capillary LC with an 11.5-T FTICR mass spectrometer for proteome analysis. EXPERIMENTAL SECTION LC Instrumentation and Materials. Two high-pressure (10 000 psi) LC pumps purchased from Isco (model 100DM, Isco, Lincoln, NE) were used for mobile-phase delivery. Passive feedback switching valves for mobile-phase switching (four-port C2XH-1004) and sample injection (six-port C2XH-1006), stainless steel tubing (1.6-mm o.d. with various inner diameters), stainless steel microvolume connectors (unions and tees), and stainless steel screens (2-µm pores) were purchased from Valco (Houston, TX). Various diameters of fused-silica capillary tubing were obtained from Polymicro Technologies (Phoenix, AZ). Various inner diameters of PEEK tubing used for column connections were purchased from Upchurch Scientific (Oak Harbor, WA). Reversedphase C18-bonded particles with 1.5-µm diameter (100-Å pore size) were obtained from Alltech (Deerfield, PA), while C18-bonded particles with 2- (100-Å pore size) and 3-µm (300-Å pore size) diameters were kindly supplied by Phenomenex (Terrence, CA). Various HPLC grade solvents were purchased from Aldrich (Milwaukee, WI). Preparation of Cytosolic Tryptic Peptides from Saccharomyces cerevisiae. S. cerevisiae (yeast) haploid strain S288C was harvested by centrifugation at early/mid-log phase (OD600 ) 3.6) from 2 L of YPD media distributed in four baffled flasks (2 L) incubated in a 37 °C/250 rpm shaker-incubator. Cell pellets (4.0 g) were washed several times in cytosol buffer [20 mM Hepes (pH 6.8), 250 mM sorbitol, 150 mM KOAc, 1 mM MgOAc, 1 mM DTT] and resuspended in 1.5 volumes of cytosol buffer. Multiple 1-mL aliquots were dispensed to 2-mL tubes, and 0.5-mm glass beads (BioSpec Products, Bartiesville, OK) were added to fill the remaining to volume of each tube. Cells were disrupted by five 30-s cycles (4600 rpm) of bead-beating (Mini Beadbeater, BioSpec Products) separated by 1-min intervals on ice. A postnuclear supernatant was prepared by centrifugation (Eppendorf 5815, 5000g, 5 min), and a cytosolic fraction was obtained by centrifugation on a TL-100 ultracentrifuge (TLS-55, 100000g at 4 °C for 30 min, Beckman-Coulter, Fullerton, CA). Cytosolic protein content (23 mg/mL) was determined using a modified Bradford assay (BioRad, Hercules, CA) using IgG (BioRad) as a standard. Yeast cytosol was diluted to 4 mg/mL in the digestion buffer [50 mM Tris (pH 8.0), 1 M urea, 2 mM CaCl2] and was denatured for 5 min at 90 °C. After cooling to 37 °C, sequencing grade modified trypsin (40 µg/mL, 1:100, w/w, Promega, Madison, WI) was added, and the sample digested overnight. The insoluble material was centrifuged (Eppendorf 5815, 17000g, 15 min), and the supernatant was redigested with additional trypsin (20 µg/ mL, overall 1:67, w/w) for 16 h at 37 °C. Sample were stored at 4 °C and recentrifuged prior to analysis.

Figure 1. Schematic diagram of the multiple-capillary LC system for operation at 10 000 psi. The system was operated at a constant pressure while the mobile-phase composition gradient was generated by switching the mobile phase directed into the mixing bomb from A to B using a four-port passive feedback switching valve. The insets show the dual-capillary device and the modified use of the passive feedback switching valve for packed capillary LC under mobile-phase gradient conditions.

Preparation of Packed Capillaries and Capillary LC/UV Experiments. Packed capillaries were made using a previously described procedure.17 Briefly, one end of the fused-silica capillary (150 µm i.d. × 360 µm o.d.) was connected to a microvolume union (Valco) using PEEK tubing (380-µm i.d.) to position a steel screen having 2-µm pores. A 0.5-µm pore inlet filter (LC packings, San Francisco, CA) was used for supporting the particles when packing capillaries with 1.5- and 2-µm particles. The other end of the capillary was connected to a stainless steel vessel, into which the bonded packing particles were introduced. Using an HPLC grade solvent mixture of acetonitrile (ACN)/H2O (90:10, v/v), the particles in the vessel were packed into the capillary by gradually increasing the pressure from 500 to 10 000 psi using an Isco pump. After packing, the capillary column was conditioned in an ultrasonic bath for 5-10 min at a constant pressure of 10 000 psi. When using a UV detector, the separation was monitored at 215 nm (Spectra 100 UV/visible detector, Spectra-Physics). A 75 µm i.d × 360 µm o.d. fused-silica capillary connection tube (2.5-cm distance from the column outlet to the detection point) was used for the detection window by burning off the capillary polyimide coating. Capillary LC-FTICR Studies. The LC capillary was coupled on-line to an 11.5-T FTICR mass spectrometer, designed and constructed at our laboratory, through an atmospheric pressure ESI source, external to the magnetic field of the spectrometer.

External accumulation23 of ions in a linear quadrupole ion trap (a segment of the spectrometer’s ion guide) was employed. This essentially converted the continuous mode ESI ion source into a pulsed ion source, which is more compatible with the sequential nature of FTICR operation. It also provides enhanced dynamic range (see later discussion) and permitted optimal sensitivity since ion accumulation can occur simultaneously with FTICR excite/ detect events. LC separations were monitored by the spectrometer through a total of ∼2000 spectra, each requiring ∼5.7 s. It should be noted that though ions were dynamically trapped in the FTICR cell, soft-gas assistance (i.e., injection of a small amount of nitrogen gas) was also used to reduce their translational energy prior to excitation and detection and improve spectrum quality. The maximum signal intensity corresponded to a procedure where ion collisional damping was performed for 5 s and was the major contributor to the total acquisition sequence during the LC-MS separation. Similar performance with reduced cooling times is feasible, pending the optimization of ion transfer and trapping conditions, and will further speed future studies. RESULTS AND DISCUSSION Strategies for Multiple-Capillary LC Operation at 10 000 psi. High-throughput and high-resolution capillary LC separations under mobile-phase gradient conditions were accomplished for proteomic applications using a multiple-column system designed Analytical Chemistry, Vol. 73, No. 13, July 1, 2001

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to eliminate time dedicated to column reequilibration (washing with mobile phase A) following each separation. Currently, multiple-column systems are seldom used for capillary LC. Likely explanations for this are difficulties associated with designs that produce extra dead volume(s) and degrade separation quality and the present lack of acceptance of capillary LC for routine analyses. However, the increased throughput provided by a multiple-column system remains attractive, and the advantages of small-diameter capillary columns become increasingly significant for proteomic and other applications where sample size is often limited. We have implemented a strategy to perform high-efficiency multiple-capillary LC separations using a single LC pumping system consisting of several dual-capillary column devices that operate at a constant pressure (Figure 1). These dual-capillary column devices are serially connected to two high-pressure mobile-phase lines, fed using independent LC pumps (mobile phase A and mobile phase B). Each dual-capillary column device (Figure 1 inset) is composed of a mobile-phase mixer (2.8 mL), a switching valve for mobile-phase gradient, a split line (6 m × 30 µm i.d. fused silica), a switching valve sample injector, and two capillary separation columns. While the constant pressure LC operation offers limited flexibility for manipulating the mobilephase gradient to optimize separation selectivity, reliably obtaining high separation efficiencies is more important for separations of highly complex samples such as from global cellular protein digestions. The only limitation to the number of such capillary column devices that can be connected in a single system results from the flow rate of the high-pressure LC pumps. For example, if each dual-capillary column device operates at a mobile-phase flow rate of ∼20 µL/min (including splitting, typically used for our capillary LC experiments), up to 10 dual-capillary column devices can be connected using pumps that supply a stable flow rate of ∼0.2 mL/min at the desired pressure. Use of Passive Feedback Switching Valves for 10 000 psi Capillary LC. The Valco passive feedback switching valves, recently developed to improve valve lifetime, are used for our highefficiency multiple-capillary LC system. When a passive feedback six-port switching valve (Cheminert model C2XL) was used as the sample injector, the relatively large volume (∼100 µL) of the passive feedback channel greatly degraded the separation quality due to an extreme delay of the gradient. For example, for a column flow rate of 1-2 µL/min that is typically used for optimal capillary LC separations with 150-µm-i.d. packed capillaries, the corresponding gradient delay time resulting from the passive feedback channel can be >50 min. The Figure 1 inset shows the modified connection arrangement used in this work with the passive feedback switching valve injector. A microvolume tee (150-µm pore size, Valco) was connected to ports 4 and b with 4 cm × 125 µm i.d. × 1.6 mm o.d. and 10 cm × 125 µm i.d. × 1.6 mm o.d. pieces of stainless steel tubing, respectively. The dead volume between the tee and the separation capillary column (at port 3) after modification is 200 switching cycles. More importantly, these valves permit quantitative sample injection and are readily amenable to automation using various commercial electronic actuators. Separation Efficiency and Reproducibility of the MultipleCapillary LC System. The high-efficiency multiple-capillary LC system consisted of several independent dual-capillary column devices of identical configuration. The separation efficiency and reproducibility of the dual-capillary LC device, which used column switching (150-µm port valve), connection tubing (15 cm × 50 µm i.d.), and two different separation capillaries, were experimentally evaluated. Figure 3 shows two consecutive separations of a

Figure 3. Separation efficiency and reproducibility realized using the dual-capillary LC arrangement illustrated in Figure 1. Conditions are identical to Figure 2A.

yeast tryptic digest using each capillary column for the dualcapillary column device. Comparing with the results obtained using single capillary LC (Figure 2A), the same separation efficiency was reproducibly obtained with both capillaries for this extremely complex sample. The average errors for elution time and peak height are 4% and 8%, respectively, for recognizable peaks with elution times of 100 000 detected putative polypeptides) support our belief that proper selection of the mobile phases is desirable for maintaining both separation and ESI efficiency. Examples illustrating the qualities of the separation and the mass spectrometry are shown in Figure 8. Figure 8A shows a typical single spectrum with insets showing exploded views of several regions of the spectrum. Since the FTICR has a practical capacity of ∼107 charges, there is a finite limit in either the number of equiabundant peaks that can be observed in a single spectrum or the maximum dynamic range in a single spectrum. On the basis of our experience, a single high-resolution FTICR spectrum provides a maximum “peak capacity” corresponding to >105 species. This indicates a vast potential for far surpassing the effective peak capacity of capillary LC-FTICR separations we previously reported, where ∼106 polypeptide species were potentially distinguishable in a single analysis.17 Figure 8B shows a very narrow range (m/z 972.515-972.535) reconstructed ion chromatogram where a number of both high- and low-abundance peaks eluted in this small m/z window during the separation. The power of the approach, however, is that this quality of information is obtained over a wide m/z range. Figure 8B also demonstrates the excellent peak shapes typically obtained. High-quality separations are of enormous importance for proteomics since the detection methodology always places the ultimate limitations on the sample complexity and/or dynamic range that can be addressed. More abundant polypeptides were typically observed to elute over three to five spectra (each scan of 5.7 s), while minor components were observed to elute over only one to two spectra. Using an average peak width at the base of 25 s, the chromatographic peak capacity (for a resolution of unity25) corresponds to ∼650 (1.5 × 180 × 60/25) under our ESI-FTICR analysis

conditions. This value is lower than that we obtained using our 3-T FTICR with a shorter spectrum acquisition time of 2.5 s, where a chromatographic peak capacity of ∼1000 was achieved.17 The combined effective resolving power supplied by the 2-D capillary LC-FTICR separation is critical when accurately measured masses (e.g., a mass measurement accuracy of 103 in a single spectrum (see below). (Even assuming that up to 10 000 peaks might originate from polymeric and solvent contaminants, we conservatively estimate that >100 000 of these components are polypeptides arising from digestion of soluble yeast proteins.) Thus, only a small fraction of the resolving power of 2-D capillary LC-FTICR is utilized for this complex yeast global tryptic digest. (25) Giddings, J. C. United Separation Science; John Wiley & Sons: New York, 1991; p 105.

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Figure 8. Resolving quality of the capillary LC and 11.5-T ESI-FTICR. Conditions are identical to Figure 7. Refer to text for details.

It must be noted that although FTICR can supply a resolving power of ∼105 species, this power by itself is not sufficient to resolve the extremely complex mixtures of cellular polypeptides. For example, ∼6000 proteins are predicted to be potentially expressed by the yeast genome,26 which can yield >350 000 different tryptic polypeptides and ∼195 000 having masses between 500 and 5000. Even if only ∼20% of the ∼6000 proteins are expressed at detectable levels under a specific set of conditions, an ideal tryptic digestion would conceivably yield ∼40 000 different (26) http://www.proteome.com/databases/YPD/YPDfacts.html.

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polypeptides, and a much larger number if modified and incompletely digested polypeptides are also considered. Many polypeptides will yield multiple charge states (e.g., 2+, 3+) following ESI, with each charge state comprising multiple isotopic peaks. Complexity of this type is illustrated in Figure 8B, which shows more than 20 peaks evident in a very narrow m/z range (0.02 Da) and having apparent retention factor27 differences of as little as 0.006, and that would be difficult to resolve using low-efficiency (27) Snyder, L. R.; Dolan, J. W. In Advances in Chromatography; Brown, P. R., Grushka, E., Eds.; Marcel Dekker: New York, 1998; Vol. 38, pp 115-187.

Figure 9. Capillary LC-FTICR 2-D display for a portion of the analysis of a yeast global soluble protein tryptic digest that contained a total of ∼110 000 detected components. Conditions are identical to Figure 7.

separations. Such complexity can result in the need for even greater FTICR resolving power and/or higher efficiency separations prior to FTICR. While FTICR resolution can be increased, typically at the cost of a slight increase in the spectrum acquisition time or magnetic field strength, along with a more significant increase in the data storage requirements, the ion trap capacity issues mentioned above still impose the greatest limits on overall dynamic range. This issue is being addressed as described below. It should be noted that high-resolution capillary LC-FTICR experiments on complicated samples produced huge data sets (>1.5 GB) for each analysis. Even using the most current desktop PC computers, nearly 2 days for data processing is required. The development of higher speed computers and the implementation of multiprocessor, PC clusters, and distributed computing platforms will be a necessary aspect of high-throughput proteome analyses. Dynamic Range of 2-D Capillary LC-FTICR Analysis. As shown by the example in Figure 10, the dynamic range obtainable in a single FTICR mass spectrum exceeds 103. The most highly abundant polypeptide eluted over 13 spectra, while low-abundance polypeptides often elute over only a single spectrum. Therefore, the effective dynamic range for detection of polypeptides can approach ∼104 if they have the same ionization or detection efficiency, just on this basis. Furthermore, if one’s aim is protein identification, then a significant (perhaps 10-fold) increase in effective dynamic range will result due to the variable electrospray ionization or detection efficiency for different polypeptide sequences. This variation in overall detection efficiency is evident in analysis of unseparated tryptic digests where polypeptide fragments differ greatly in their intensity compared to their nominally expected equimolar abundances. More important, however, is the ion accumulation process used with FTICR. Ion

introduction from ESI, transfer through the ion funnel, and “external” ion accumulation are more efficient when ion production rates are lower. Although we have not yet quantified this effect, it is clear that sensitivity is greatly improved for low-abundance peaks that are chromatographically separated from high-abundance peaks. This contribution likely accounts for at least a 1 order of magnitude increase in dynamic range and can potentially be much greater if ion bias effects that result from “overfilling” of the ion accumulation region can be mitigated.28 Upon consideration of these factors we estimate the dynamic range currently achieved is approximately 104-105 and believe that a 1 order of magnitude further gain is achievable if unbiased ion accumulation can be achieved for greater than the 100-ms periods used in this work. In this regard, it should be noted that continuous external accumulation of ions between transfers to the FTICR trap would increase the ion accumulation greatly, potentially providing an equivalent gain in dynamic range if the quadrupole accumulation region was not overfilled, and could potentially increase the dynamic range of FTICR analyses to >106. Our current efforts are aimed at achieving such an extended dynamic range. As can be seen from the above discussion, the dynamic range in a single FTICR mass spectrum is limited by both the charge capacities of the “external” ion accumulation region and of the FTICR cell. In practice, the useful charge capacity of the external accumulation quadrupole trap is on the order of 107 charges if undesirable effects due to overfilling are to be avoided. These combined effects include bias due to charge stratification in the accumulation quadrupole and “coalescence” of closely spaced m/z ion packets in the FTICR trap. Improvements in the ESI source design and use of an electrodynamic ion (28) Belov, M.; Nikolaev, E. W.; Alving, K.; Smith, R. D., submitted.

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Figure 10. Effective dynamic range of a single FTICR mass spectrum from a capillary LC-FTICR analysis of a global yeast soluble protein tryptic digest obtained under capillary LC data acquisition conditions. Conditions are identical to Figure 7.

funnel now allow currents of >1 nA of analytically useful ions to be transmitted to the ion accumulation regions. This corresponds to ∼1010 charges/s, a factor of ∼103 in excess of the ion population than can currently be analyzed by FTICR in a single spectrum (even if only a 20% transfer efficiency is assumed with the present 11.5-T instrument). Our efforts are thus directed toward establishing a routinely useful “active dynamic range enhancement” capability, in which the information from a proceeding spectrum is used to remove the high-abundance species in an rf-only quadrupole just prior to the ion accumulation quadrupole region.24,28,29 In this fashion, every other spectrum would “dig deeper” into the proteome and provide more information on lower abundance species. While the overall dynamic range potentially achievable with this approach should significantly exceed 106, its practical utility remains to be fully demonstrated. Protein Identification. The analysis of 2-D capillary LC-FTICR data for protein identification will be reported in detail elsewhere.30 Briefly, the detected isotopic distributions were deconvoluted using the ICR-2LS software package31 developed at our laboratory, and the resultant neutral molecular masses were then visualized in as a 2-D display (Figure 9) using also in-house-developed software.32 These software packages comprise a spectrum of custom data processing/analysis tools and database searching capabilities. We have used LC-FTICR multiplexed MS/MS data30 to enhance the mass measurement accuracy of 11.5 T by the use (29) Harkewicz, R.; Belov, M. E.; Anderson, G. A.; Pasˇa-Tolic´, L.; Masselon, C. D.; Prior, D. C.; Udseth, H. R.; Smith, R. D., submitted. (30) Li, L.; et al., manuscript in preparation. (31) 31.ICR2LS; Anderson, G. A., Bruce, J. E., Eds.; Pacific Northwest National Laboratory: Richland, WA, 1995. (32) 32.LaV2DG; Tolic´, N., Ed.; Pacific Northwest National Laboratory, Richland, WA, 1999.

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of “lock masses” (i.e., known species that serve as effective internal calibrants) derived from a set of commonly occurring and highly abundant species. The resultant ∼110 000 unique isotopic distributions include many that can serve as accurate mass tags (AMTs), i.e., peptide masses measured with high enough MMA such that its mass is unique among all of the possible peptides predicted from a genome. The masses for the detected yeast polypeptides were searched against the yeast protein database (obtained from ftp://ncbi.nlm.nih.gov/genbank/genomes/S_cerevisiae/), yielding “hits” for >9000 peptides corresponding to ∼1000 proteins predicted from unique yeast open reading frames, for only the soluble protein. The capability to model elution times (or elution order) of polypeptides and their structures (hydrophobic properties) should extend the number of identifications. In addition, as we will report elsewhere, an initial validation of AMTs based upon MS/MS serves to provide an even more conclusive approach to peptide identifications without sacrificing the desired throughput in the subsequent application of AMTs for protein identification in quantitative perturbation studies. CONCLUSIONS Instrumentation for improved proteomic measurements has been developed and demonstrated. Passive feedback switching valves (both four- and six-port) could be effectively used for 10 000 psi capillary LC separations with lifetimes exceeding 4 months and 200 switching cycles. These valves made it possible to implement a high-efficiency multiple-capillary LC system equipped with small particle packed long capillary columns (e.g., 85-cm capillaries packed with 3-µm particles). Compared with a singlecapillary LC, in which the separation capillary was directly connected to the sample injector, the described dual-capillary

column device, in a multiple-capillary LC system, showed no separation efficiency loss under the identical mobile-phase gradient conditions. The capillary columns could be regenerated within the time period (∼3 h) required for the parallel, high-efficiency separation of a peptides resulting from a complex global yeast proteolytic digest on the second column of the dual-column device. Combining the high-efficiency capillary LC system with a high magnetic field (11.5 T) FTICR for 2-D LC-FTICR analysis provided a resolving power capability (peak capacity) of ∼6 × 107. Large numbers of putative polypeptides (>100 000) were reproducibly observed from a single global yeast digest LC-FTICR analysis that lasted only 3-4 h. We estimate the present dynamic range of our FTICR analysis to be ∼105 and believe this can potentially be extended to >107. In future publications, we will describe the (33) Conrads T. P.; Alving, K.; Veenstra, T. D.; Belov, M. E.; Anderson, G. A.; Anderson, D. J.; Lipton, M. S.; Pasˇa-Tolic´, L.; Udseth, H. R.; Chrisler, W. B.; Thrall, B. D.; Smith, R. D. Anal. Chem. 2001, 73, 2132-2139.

methodologies being developed for high-throughput protein identification and, in combination with stable-isotope labeling,33 for sensitive and quantitative proteome-wide measurements. ACKNOWLEDGMENT The authors thank Dr. Jeff Layne of Phenomenex (Torrance, CA) for the generous supply of 3- and 2-µm C18 particles. The research was largely supported by the National Cancer Institute under Grant CA 81654; other portions of the research were supported by the Office of Environmental and Biological Research, U.S. Department of Energy. Pacific Northwest National Laboratory is operated by Battelle Memorial Institute for the U.S. Department of Energy through Contract DE-ACO6-76RLO 1830. Received for review November 29, 2000. Accepted April 16, 2001. AC001393N

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