Anal. Chem. 2004, 76, 2734-2740
Protein Characterization by On-Line Capillary Isoelectric Focusing, Reversed-Phase Liquid Chromatography, and Mass Spectrometry Feng Zhou and Murray V. Johnston*
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716
Two-dimensional polyacrylmide gel electrophoresis (2DPAGE), perhaps the most widely used method in proteomics research, is often limited by sensitivity and throughput. Capillary isoelectric focusing (CIEF) coupled with electrospray ionization (ESI) mass spectrometry (MS) provides a liquid-based alternative to 2D-PAGE that can overcome these problems but is limited by ampholyte interference and signal quenching in ESI-MS. Inserting a reversed-phase liquid chromatography (RPLC) step between CIEF and MS can remove this interference. In this work, a CIEF-RPLC-MS system is described for separation and characterization of proteins in complex mixtures. CIEF is performed with a microdialysis membrane-based cathodic cell that also permits protein fractions to be collected, washed to remove ampholyte, and analyzed by RPLC-MS. CIEF performance with this cell is equivalent to that achieved with a conventional cathodic cell, and no loss of protein is observed during faction collection. The cell can be easily and safely retrofitted into commercial instrumentation and is applicable for peptide analysis as well. Protein detection at the low-femtomole level is demonstrated with little or no interference from ampholyte, and CIEF-RPLC-MS data are used to construct a plot of pI vs MW for a protein mixture. The current instrumental configuration allows seven fractions in the pI range 3-10 to be analyzed by RPLC-MS in 2 h. High-throughput DNA sequencing has led to a rapid expansion of genomic databases. While a genome is relatively static, the corresponding proteome is dynamic and not as well characterized.1 Protein expression levels can change quickly and do not correlate well with mRNA levels. Individual proteins are modified and processed in ways that are not predicted by the DNA sequence. Characterizing these variations is important because they define and control cellular processes.2 In a proteomic sample, there are usually a large number of proteins which vary widely in physicochemical properties including molecular weight (MW), isoelectric point (pI), solubility, acidity/basicity, and hydrophobicity/hydrophilicity. Moreover, the protein concentrations can extend over 6 orders of magnitude, and it is often the least abundant proteins * To whom correspondence should be addressed. Phone: (302) 831-8014. Fax: (302) 831-6335. E-mail:
[email protected]. (1) Pandey, A.; Mann, M. Nature 2000, 405, 837-846. (2) Hille, J. M.; Freed, A. L.; Watzig, H. Electrophoresis 2001, 22, 4035-4052.
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that are of the greatest interest in systems biology. For these reasons, there is a continuing need to develop and evaluate new platforms for protein separation and analysis.3 Two-dimensional polyacrylmide gel electrophoresis (2D-PAGE) is the core technology for proteomic studies.4 As a multidimensional separation method, 2D-PAGE can resolve >1000 proteins in a single run.5 However, two main drawbacksssensitivity and throughputsultimately limit its use in proteomics research. 2DPAGE usually employs coomassie blue or silver staining to visualize separated proteins in the gel. Silver staining is the more sensitive of these, detecting 0.1-10-ng amounts of protein, which corresponds to 20-200 fmol for a 50-kDa protein.3,4,6 The dynamic range of 2D-PAGE is at best 104, making it difficult to study lowabundance proteins.6 Throughput is hindered in that the entire process of gel preparation to protein separation and analysis is slow and difficult to fully automate.1,7 In principle, multidimensional liquid-based separations could overcome these problems. Liquidbased methods facilitate direct coupling with mass spectrometry (MS) for protein characterization. If the separation mode is analogous to 2D-PAGE, then comprehensive and powerful databases built from 2D-PAGE data can be effectively utilized.2 Capillary isoelectric focusing (CIEF) coupled with MS is a promising approach to this problem.8-15 CIEF provides a highresolution separation based on pI, and MS provides a mass measurement with high precision and accuracy. A display of pI versus MW can be obtained from CIEF-MS data with higher (3) Shen, Y.; Smith, R. D. Electrophoresis 2002, 23, 3106-3124. (4) Gorg, A.; Obermaier, C.; Boguth, G.; Harder, A.; Scheibe, B.; Wildgruber, R.; Weiss, W. Electrophoresis 2000, 21, 1037-1053. (5) O’Farrel, P. H. J. Biol. Chem. 1975, 25, 4007-4021. (6) Corthals, G. L.; Wasinger, V. C.; Hochstrasser, D. F.; Sanchez, J. C. Electrophoresis 2000, 21, 1104-1115. (7) Manabe, T. Electrophoresis 1999, 20, 3116-3121. (8) Tang, Q.; Harrata, A. K.; Lee, C. S. Anal. Chem. 1995, 67, 3515-3519. (9) Yang, L.; Lee, C. S.; Hofstadler, S. A.; Smith, R. D. Anal. Chem. 1998, 70, 4945-4950. (10) Jensen, P. K.; Paa-Tolic, L.; Anderson, G. A.; Horner, J. A.; Lipton, M. S.; Bruce, J. E.; Smith, R. D. Anal. Chem. 1999, 71, 2076-2084. (11) Jensen, P. K.; Paa-Tolic, L.; Peden, K. K.; Martinovic´, S.; Lipton, M. S.; Anderson, G. A.; Tolic, N.; Wong, K-.K.; Smith, R. D. Electrophoresis 2000, 21, 1372-1380. (12) Paa-Toli, L.; Jensen, P. K.; Anderson, G. A.; Lipton, M. S.; Peden, K. K.; Martinovi, S.; Toli, N.; Bruce, J. E.; Smith, R. D. J. Am. Chem. Soc. 1999, 121, 7949-7950. (13) Martinovic, S.; Berger, S. J.; Pasa-Tolic, L.; Smith, R. D. Anal. Chem. 2000, 72, 5356-5360. (14) Liu, T.; Shao, X. X.; Zeng, R.; Xia, Q. C. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2002, 34, 423-432. (15) Clarke, N. J.; Naylor, S. Biomed. Chromatogr. 2002, 16, 287-297. 10.1021/ac035446n CCC: $27.50
© 2004 American Chemical Society Published on Web 04/14/2004
throughput and sensitivity than 2D-PAGE.10 However, substantial problems remain. Most important is the need for ampholyte in CIEF, which coelutes with the analyte and perturbs the electrospray ionization (ESI) process in a manner that suppresses analyte signal intensity and degrades mass resolution.8,16-17 In addition, the resolution of CIEF alone is not sufficient for complete separation of complex samples. When multiple proteins are coeluted into an ESI source, some of them will be subject to additional signal suppression by the presence of others,18 which increases the detect limit and dynamic range. Finally, it is difficult to add reagents that enhance solubility of hydrophobic proteins because their presence, like ampholyte, degrades ESI performance. For these reasons, it is desirable to insert an additional separation step between CIEF and MS, for example, reversedphase liquid chromatography (RPLC). RPLC is easily coupled with ESI-MS and provides the opportunity to remove ampholyte and further separate proteins that are partially separated by CIEF. A CIEF-RPLC-MS system for peptides has been reported by Lee and co-workers.19 In this system, an injection loop attached to a microselection valve is placed between the anodic and cathodic cells to transfer peptide fractions into the RPLC. Ampholyte is removed from the peptide fraction with a C18 trap column prior to RPLC. This system is capable of detecting thousands of peptides in a single run with high sensitivity. In the present work, an improved CIEF-RPLC-MS system is evaluated for protein separation and characterization. A microdialysis membrane separates the cathodic cell from the separation capillary. During the focusing step, catholyte is able to traverse the membrane but protein molecules cannot. After focusing, proteins are hydrodynamically pushed past the membrane to a microselection valve that collects and transfers fractions to the RPLC for further separation. This arrangement permits a linear pH gradient to be maintained in the separation capillary with no voltage on the microselsection valve. The cathodic cell and microselection valve can be easily and safely retrofitted into commercial instrumentation. Protein mixtures can be separated and analyzed in 2 h with high sensitivity and resolving power. EXPERIMENTAL SECTION Reagents and Materials. Phosphoric acid, sodium hydroxide, and HPLC grade acetonitrile and acetic acid were obtained from Fisher Scientific (Fairlawn, NJ). Ampholyte (eCap cIEF 3-10) was obtained from Beckman Coulter, Inc. (Fullerton, CA). Ribonuclease A from bovine pancreas, cytochrome c from bovine heart, myoglobin from horse heart, carbonic anhydrase II from bovine erythrocytes, insulin from bovine pancreas, β-lactoglobulin from bovine milk, trypsin inhibitor type I-S from soybean, and [Glu′]fibrinopeptide B were obtained from Sigma (St. Louis, MO). Bovine serum albumin was obtained from Roche (Basel, Switzerland). CCK flanking peptide was obtained from Beckman Coulter, Inc. Deionized water was obtained from a Millipore (Bedford, MA) Simplicity 185 system. All buffers were filtered through a Millipore Millex GP filter (0.22 µm) before use. PEEK tubing was obtained from Alltech Associates, Inc. (Deerfield, IL) (16) Tang, Q.; Harrata, A. K.; Lee, C. S. Anal. Chem. 1996, 68, 2482-2487. (17) Tang, Q.; Harrata, A. K.; Lee, C. S. Anal. Chem. 1997, 69, 3177-3182. (18) Sterner, J. L.; Johnston, M. V.; Nicol, G. R.; Ridge, D. P. J. Mass Spectrom. 2000, 35, 385-391. (19) Chen, J.; Balgley, B. M.; DeVoe, D. L.; Lee, C. S. Anal. Chem. 2003, 75, 3145-3152.
Figure 1. Diagram of the microdialysis membrane-based cathodic cell.
Construction of a Microdialysis Membrane-Based Cathodic Cell. A schematic of the microdialysis membrane-based cathodic cell for CIEF is shown in Figure 1. The cathodic cell is built around a PEEK tee (Valco Instruments Co. Inc., Houston, TX). One collinear port is connected to a 55-cm CIEF capillary (eCAP neutral capillary; 50-µm i.d., 375-µm o.d.; Beckman Coulter). The opposite collinear port is connected to a 20-cm fused-silica capillary that leads to a waste vial. The perpendicular port is connected to the cathodic vial. The cathodic vial is built from two reducing unions and a 5-cm length of Dayco polyethylene tubing (1/4-in, o.d., 3/8-in. i.d.). The first reducing union connects the polyethylene tubing to the PEEK tee through a 3-cm length of PEEK tubing (1/16-in. o.d., 400-µm i.d.) that attaches to the perpendicular port. A microdialysis membrane (MW cutoff 3500; Spectrum laboratory, Rancho Domingnes, CA) cut to a 1/16-in. diameter is inserted into the perpendicular port and supported by a 0.5-µm stainless steel filter. The second reducing union connects the other end of polyethylene tubing to another 3-cmlength PEEK tubing (1/16-in. o.d., 400-µm i.d.) containing a 6-cm Pt wire as the cathodic electrode. This cathodic “vial” is filled with ∼1.5 mL of catholyte after degassing. Any bubbles in cathodic vial should be carefully removed. CIEF with the Microdialysis Membrane-Based Cathodic Cell. CIEF is performed with the microdialysis membrane-based cathodic cell mounted inside a commercial automated capillary electrophoresis system (Beckman P/ACE MDQ with a UV detector). The CIEF capillary is filled with ampholyte solution (1: 80, ampholye/water). Protein/peptide test mixtures are injected from anodic side for 2 min by pressure. Focusing is performed in an electric field of 300 V/cm; the anolyte is 91 mM H3PO4 and the catholyte (sealed in the cathodic cell) is 20 mM NaOH. The Pt wire in cathodic cell is connected to the negative electrode of the instrument and set to 0 V. After a focusing period of 4 min, 0.8 psi pressure is applied to the anodic end of the capillary to hydrodynamically push the focused bands through a UV absorption detector located at 10 cm upstream of the cathodic cell. The 300 V/cm electric field is maintained across the capillary during hydrodynamic mobilization. Absorption is monitored at 280 nm. Sample losses to the cathodic cell are studied by repositioning the UV detector 10 cm downstream of the cathodic cell. All capillary lengths and sizes are identical for each detector location. CIEF-RPLC-MS. A diagram of the entire CIEF-RPLC-MS system is shown in Figure 2. The membrane-based cathodic cell assembly is connected to a six-port electronically controlled Analytical Chemistry, Vol. 76, No. 10, May 15, 2004
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Figure 2. Diagram of the CIEF-RPLC-MS system.
microselection valve (Valco part no. C2NH-4096EH;) with a 5-cmlength fused-silica capillary (50-µm i.d., 375-µm o.d.; Polymicro, Phoenix, AZ). The ampholyte solution (1:80, ampholye/water) and protein/peptide test mixtures are injected and separated by CIEF as described above. The focused bands are then hydrodynamically pushed past the cathodic cell into a 1-µL stainless steel injection loop (Valco part no. CSLN1K) attached to the microselection valve. The valve position is then switched, and proteins fractioned by loop are transferred to a C18 reversed-phase trap column (300-µm i.d., 5-mm length; LC-Packing, San Francisco, CA) attached to a second six-port valve located on a commercial capillary LC system (Waters, Milford, MA). Figure 2 illustrates the valve positions during the transfer step. After trapping, both six-port valves are switched back to their original positions. Fractioned proteins are eluted from the trap column and separated by RPLC. Meanwhile, another fraction is collected on the 1-µL loop and the sequence is repeated. During the entire time, the 300 V/cm electric field is applied to the CIEF capillary to maintain isoelectric focusing. In practice, it is found that some electroosmotic flow occurs from the anodic end of the CIEF capillary during the course of the experiment. This flow is counterbalanced hydrodynamically by placing the waste vial ∼10 cm above the anodic end of CIEF capillary. The protein fraction sampled by the injection loop is loaded on trap column and washed with a solution of 94.9% H2O, 5% acetonitrile and 0.1% acetic acid at a flow rate of 20 µL/min for 3 min. Most ampholyte is removed during this time. The protein fraction is then eluted from trap column by mobile phase and further separated on a C4 reversed-phase column (300-µm i.d., 5-cm length, Microm, San Jose, CA). Mobile phase A (94.9% H2O, 2736
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5% acetonitrile, and 0.1% acetic acid) and mobile phase B (94.9% acetonitrile, 5% H2O, and 0.1% acetic acid) are delivered at a flow rate of 20 µL/min using a two-step gradient of 5 (phase B from 25 to 60%) and 2 min (phase B from 60 to 90%). For the proteins/ peptide considered in this study, no sample carryover from one fraction to the next was observed and each compound was observed in the same fraction over several hundred runs. Proteins are mass analyzed with a Q-TOF Ultima API-US mass spectrometer (Micromass, Manchester, U.K.). The sample eluted from the RPLC column is sent into a conventional API-ESI source. The ESI voltage is 2.5 kV; the mass spectrum is acquired from 600 to 2000 Da with a scan time of 0.5 s and an interscan time of 0.1 s. Data processing is performed with MaxEnt1, which converts the raw spectrum of multiply charged ions into a deconvoluted display of singly charged ions. The pI and MW of each protein obtained from CIEF and MS analysis respectively are then visualized in a format similar to 2D-PAGE. RESULTS AND DISCUSSION CIEF Performance. Figure 3a shows the separation achieved with the microdialysis membrane-based cathodic cell for a sample containing 0.1 nmol of ribonuclease A, myoglobin, carbonic anhydrase II, β-lactoglobulin, trypsin inhibitor, and 1 nmol of CCK flanking peptide. For this experiment, the absorbance detector was placed upstream of the cathodic cell as described in the Experimental Section. The current profile in Figure 3b shows a rapid decrease in the first few minutes with little change afterward. A slight upturn in current is observed at very long times and is caused by hydrodynamic movement of phosphoric acid into the capillary. The current profile in Figure 3b is virtually identical to
Figure 3. CIEF separation with the microdialysis membrane-based cathodic cell: (a) UV absorption (280 nm) vs time; (b) current profile.
Figure 4. Calibration curve of pI vs migration time for CIEF with the microdialysis membrane-based cathodic cell.
that obtained from a separation using the commercially supplied cathodic cell, which indicates free movement of hydroxide ions across the microdialysis membrane. A plot of pI versus migration time with the membrane-based cathodic cell (Figure 4) is also quite similar to that obtained with the commercial cathodic cell. The slight nonlinearity observed in Figure 4 is common, and its origin has been discussed in detail previously.20 While hydroxide ions can move across the microdialysis membrane by diffusion as well as electrophoresis, diffusion is slow and a single fill of the cathodic cell can be used for several hundred runs over a time period greater than one month. Sample loss to the membrane-based cathodic cell was studied by comparing the peak area of ribonuclease A when the UV (20) Shimura, K.; Zhi, W.; Matsumoto, H.; Kasai, K. Anal. Chem. 2000, 72, 47474757.
detector was located upstream versus downstream of the cell (see Experimental Section). The position of the detector did not affect the flow rate because equal length and diameter capillaries were used in each experiment. While the peak profile was significantly broader when the detector was positioned downstream of the cell (focusing cannot be maintained as no electric field is applied downstream of the cell), no change in peak area was observed between the two detector locations within experimental error ((6%). This result suggests that little if any sample loss occurs to the membrane-based cathodic cell, which is not surprising given that the molecular weight cutoff of the membrane is much lower than the protein molecular weight. In practice, it is found that peptides with molecular weights smaller than the cutoff are also efficiently transported past the membrane. For example, when 1 pmol of [Glu′]-fibrinopeptide B is injected into the CIEF capillary, focused, transported to the sampling loop, and analyzed by RPLC-MS, a mass spectrum with signal/noise ratio higher than 3500 is obtained (data not shown). During hydrodynamic mobilization, the proteins/peptides passing by the cathodic cell are neutral and there is no significant driving force other than diffusion to extract them from the liquid stream into the cathodic cell. Hydrodynamic flow into the cathodic cell is eliminated because the cell is sealed. Stop and Go Mode. To sequentially collect and analyze pI fractions, hydrodynamic flow must be cycled on and off. In the “go” mode, pressure is applied to the anodic end to push focused bands into the injection loop. In the “stop” mode, the pressure is removed so that the analytes remaining in the capillary do not move. After the collected fraction has been transferred to the trapping column and washed, the microselection valves switch back to their original configuration and pressure is reapplied to the anodic end to collect a new fraction. While the new fraction is being collected, the fraction loaded on the trap column is eluted and analyzed by RPLC-MS. Figure 5a shows the UV absorbance trace of a CIEF separation in the stop and go mode for a sample similar to that in Figure 3, except that the concentration of Analytical Chemistry, Vol. 76, No. 10, May 15, 2004
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Figure 5. CIEF separation in the stop and go mode: (a) UV absorption (280 nm) vs time; (b) current profile. Go cycles are bracketed.
Figure 6. (a) ESI mass spectrum of 2 µM cytochrome c with 1:80 ampholyte in 50% H2O, 49% methanol, and 1% acetic acid; (b) ESI mass spectrum of 2 µM cytochrome c without ampholyte in the same buffer as (a); (c) ESI mass spectrum of 2 pmol of cytochrome c injected and analyzed by CIEF-RPLC-MS: (d) ESI mass spectra of 2 fmol of cytochrome c injected and analyzed by CIEF-RPLC-MS.
ribonuclease A (first peak) is a factor of 2 smaller. Go cycles are bracketed for clarity. Each go cycle lasts 2 min with 0.9 psi applied on anodic end of the capillary. Each stop cycle lasts 10 min during which no pressure is applied. In all, there are six cycles. Prior to stop and go cycling, the sample is focused for 4 min and then mobilized with 0.9 psi pressure for 9 min to bring the focused 2738 Analytical Chemistry, Vol. 76, No. 10, May 15, 2004
bands close to the detector. The peak widths in Figure 5a are similar to those in Figure 3, indicating that focusing is maintained during the stop cycles and resolution is not significantly degraded. Additional spikes in this plot are observed when the pressure changes. The current profile in Figure 5b, and the plot of pI versus migration time (data not shown), are similar to their counterparts
Figure 7. RPLC-MS of pI fractions. (A-G) represent pI ranges of 9.2-8.1, 8.1-7.0, 7.0-5.9, 5.9-4.8, 4.8-3.7, 3.7-2.6, and 2.6-1.5, respectively. Left panel, extracted mass chromatogram of each protein in the fraction; middle panel, raw spectrum of each protein averaged over top 80% of the LC peak; right panel, deconvoluted spectrum (singly charged) by MaxEnt1. Only the total ion chromatogram is shown for fraction F; no deconvoluted spectrum is shown for fraction G.
in Figures 3 and 4, further illustrating the effectiveness of CIEF separation under stop and go conditions. It should be noted that the mobilization pressures are different in Figures 3a and 5a, and therefore, the peak areas are also slightly different. Protein Mixture Analysis by CIEF-RPLC-MS. The fractions collected by stop and go CIEF are washed on the trap column to remove ampholyte. Figure 6 shows how ampholyte can substantially degrade protein detection by MS. Panels a and b of Figure 6 show the mass spectrum of 2 µM cytochrome c in 50% H2O, 49% methanol, and 1% acetic acid buffer with and without 1:80 ampholyte. In the presence of ampholyte, no cytochrome c signal is observed, highlighting the need to remove ampholyte prior to MS analysis. Panels c and d of Figure 6 show the mass spectra of 2 pmol and 2 fmol, respectively, of cytochrome c loaded on the capillary and analyzed by CIEF-RPLC-MS. In each case, mass spectra are obtained with little interference from ampholyte. The high sensitivity demonstrated in Figure 6d may be further improved by optimizing the RPLC step.
A mixture containing 0.1 pmol of ribonuclease A, cytochrome c, myoglobin, insulin, and β-lactoglobulin, 20 pmol of carbonic anhydrase II and bovine serum albumin, and 1 pmol of CCK flanking peptide was also analyzed by CIEF-RPLC-MS. The proteins/peptide were focused by CIEF and then divided into seven pI fractions by stop and go operation. Figure 7 shows the RPLC-MS analysis of these fractions. The labels A-G represent the 7 pI fractions analyzed. Two fractions (A and D) contained two proteins, one fraction (F) contained no proteins/peptides, and the remaining fractions contained just one protein/peptide. Three separate plots are shown for each protein/peptide: an extracted ion chromatogram, a raw mass spectrum averaged over the top 80% of the peak in the extracted ion chromatogram, and the Maxent1 deconvoluted spectrum. For fraction F, just a total ion chromatogram is shown as the only signal observed is due to residual ampholyte. For proteins smaller than 20 kDa in molecular mass, 100 fmol is generally sufficient to obtain a high-quality spectrum. Larger proteins require more sample because the ion Analytical Chemistry, Vol. 76, No. 10, May 15, 2004
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Figure 8. pI vs MW from CIEF-RPLC-MS data.
signal is distributed over many m/z values. Figure 8 shows a visual display of pI versus MW that is comparable to 2D-PAGE analysis. Molecular weights are determined from the deconvoluted spectra in Figure 7. The pI range of each fraction is estimated from the time period over which the injection loop is filled by assuming a linear relationship between migration time and pI. Since linearity is only an approximation (see Figure 4), proteins/peptides at the pI extremes appear to fall into incorrect fractions: ribonuclease A (pI ) 9.45) in the fraction having a nominal pI range of 9.2-8.1 and CCK flanking peptide (pI ) 2.75) in the fraction having a nominal pI range of 2.6-1.5. Use of a more precise calibration between pI and migration time will overcome this problem. The total time required for a single CIEF-RPLC-MS experiment with seven fractions collected is 120 min, which compares favorably with ∼12 h for 2D-PAGE separation plus additional time for spot visualization and analysis. The theoretical peak capacity of CIEF-RPLC-MS is given by21
N(CIEF-RPLC-MS) ) N(CIEF)N(RPLC)N(MS)
where N(CIEF) is 7 (given by the number of fractions), N(RPLC) is ∼10 (limited by the short separation time between the collection of successive fractions), and N(MS) is ∼15 000 (based on M/∆M (21) Giddings, J. C. Unified Separation Science; John Wiley and Sons: New York, 1991; pp 126-128.
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(50% peak height) >6000 fwhm in the deconvoluted spectrum), giving a total value of N(CIEF-RPLC-MS) greater than 106. As discussed previously, the sensitivity of CIEF-RPLC-MS is also high. While these figures of merit for CIEF-RPLC-MS are quite good, two important caveats should be kept in mind. First, the abort rate for CIEF in the stop and go mode was frequent in these initial experiments. System shutdown (initiated by the commercially supplied software during long stop cycles) is attributed to leakage current even though no such current is observed by independently. This problem it is observed can be addressed in part by modifying the apparatus to utilize short stop cycles. Second, ion signal suppression is prevalent for protein mixtures,18 so the number of proteins that can be analyzed in a single run is largely determined by the number of proteins that can be separated by CIEF and RPLC. This second problem can be addressed in several ways. Proteins can be prefractionated into wide pI ranges with a method such as the Rotofor,22 followed by successive CIEF separations over more narrow pI ranges. Monolithic columns or narrow-bore columns may provide greater separation efficiency and detection sensitivity by RPLC.23 The dynamic range for the current setup, ∼1000, is limited by the capacity of the RPLC column used. In this study, MS was used only for high-accuracy molecular mass measurement. By incorporating “top-down” strategies to characterize protein molecular ions by MSMS,24-28 proteins may be positively identified and structural features such as posttranslational modifications determined on the fly. In this manner, the entire process of protein separation and analysis could be performed in a single, automated high-throughput experiment that is directly compatible with the rather large knowledge base derived from conventional 2D-PAGE analysis. ACKNOWLEDGMENT This material is based on work supported by the National Science Foundation under Grant DBI-0096578. Received for review December 8, 2003. Accepted February 16, 2004. AC035446N (22) Shang, T. Q.; Ginter, J. M.; Johnston, M. V.; Larsen, B. S.; McEwen, C. N. Electrophoresis 2003, 24, 2359-2368. (23) Moore, R. E.; Licklider, L.; Schumann, D.; Lee, T. D. Anal. Chem. 1998, 70, 4879-4884. (24) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (25) Senko, M. W.; Beu, S. C.; McLafferty, F. W. Anal. Chem. 1994, 66, 415418. (26) Horn, D. M.; Ge, Y.; McLafferty, F. W. Anal. Chem. 2000, 72, 4778-4784. (27) Meng, F.; Cargile, B. J.; Miller, L. M.; Forbes, A. J.; Johnson, J. R.; Kelleher, N. L. Nat. Biotechnol. 2001, 19, 952-957. (28) Reid, G. E.; McLuckey, S. A. J. Mass Spectrom. 2002, 37, 663-675.
