Capillary Isoelectric Focusing-Based Multidimensional Concentration

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Anal. Chem. 2003, 75, 3145-3152

Capillary Isoelectric Focusing-Based Multidimensional Concentration/Separation Platform for Proteome Analysis Jinzhi Chen,† Brian M. Balgley,‡,| Donald L. DeVoe,§,⊥ and Cheng S. Lee*,†,⊥

Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, College of Life Sciences, Mass Spectrometry Facility, University of Maryland, College Park, Maryland 20742, Department of Mechanical Engineering and Institute for System Research, University of Maryland, College Park, Maryland 20742, and Calibrant Biosystems, 7507 Standish Place, Rockville, Maryland 20855

An integrated proteome concentration/separation approach involving on-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed for providing significant analyte concentration and extremely high resolving power toward protein and peptide mixtures. Upon completion of analyte focusing, the self-sharpening effect greatly restricts analyte diffusion and contributes to analyte stacking in narrowly focused bands with a concentration factor of ∼240. In addition to analyte focusing, CIEF as the first separation dimension resolves proteins/ peptides on the basis of their differences in pI and offers greater resolving power than that achieved in strong cation exchange chromatography. The grouping of two highly resolving and completely orthogonal separation techniques of CIEF and CRPLC, together with analyte focusing and concentration, significantly enhances the dynamic range and sensitivity of conventional mass spectrometry toward the identification of low-abundance proteins. The CIEF-based multidimensional separation/concentration platform enables the identification of a greater number of yeast soluble proteins than methods presented in the literature, yet requires a protein loading of only 9.6 µg. This protein loading is 2-3 orders of magnitude lower than those employed by the reported non-gel-based proteome techniques. The distribution of a codon adaptation index value for identified yeast proteins approximates to that predicted for the entire yeast proteome and supports the capability of CIEF-based proteome separation technology for achieving comprehensive proteome analysis. By reducing the inner diameter of chromatography columns from 180 µm to 100 µm, the required protein loading is further decreased from 9.6 µg to 960 ng, illustrating the potential usage of this proteome technology for the analy* To whom correspondence should be addressed. Phone: (301) 405-1020. Fax: (301) 314-9121. Email: [email protected]. † Department of Chemistry and Biochemistry, University of Maryland. ‡ College of Life Sciences, Mass Spectrometry Facility, University of Maryland. § Department of Mechanical Engineering and Institute for System Research, University of Maryland. ⊥ Calibrant Biosystems. | Current address: Calibrant Biosystems, 7507 Standish Place, Rockville, MD 20855. 10.1021/ac034014+ CCC: $25.00 Published on Web 05/10/2003

© 2003 American Chemical Society

sis of protein profiles within small cell populations or limited tissue samples. In the era of systems biology, computational and high throughput experimental biological approaches are combined to provide global snapshots of entire genomes and proteomes under cell-, tissue-, and disease-specific conditions.1,2 The vast number of proteins present in the proteome of a typical organism requires that separations be performed on the mixture prior to introduction into the mass spectrometer. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is still the method of choice for separating thousands of proteins in a single run3-6 while offering a “differential display” of protein expression. However, many important regulatory proteins, which are expressed at extremely low levels, are precluded in the combined 2D-PAGE-MS technique unless extensive fractionation of large quantities of protein together with the processing of a large number of narrow-range gels is performed.7 Furthermore, the 2-D PAGE/MS approach remains lacking in proteome coverage (for proteins having extreme isoelectric points or molecular masses, as well as for membrane proteins), dynamic range, sensitivity, and throughput.7,8 Consequently, considerable efforts are being devoted to the development of non-gel-based proteome technologies through the combination of various chromatography and electrokinetic separation methods with MS or tandem MS analysis.9-25 These liquid(1) Persidis, A. Nature Biotechnol. 1998, 16, 393. (2) Hood, L. J. Proteome Res. 2002, 1, 399. (3) Klose, J.; Kobalz, U. Electrophoresis 1995, 16, 1034. (4) Jungblut, P.; Thiede, B.; Zimny-Arndt, U.; Muller, E.-C.; Scheler, C.; Wittmann-Liebold, B.; Otto, A. Electrophoresis 1996, 17, 839. (5) Rabilloud, T. Anal. Chem. 2000, 72, 48A. (6) Righetti, P. G.; Castagna, A.; Herbert, B. Anal. Chem. 2001, 73, 320A. (7) Gygi, S. P.; Corthals, G. L.; Zhang, Y.; Rochon, Y.; Aebersold, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9390. (8) Smith, R. D. Nature Biotechnol. 2000, 18, 1041. (9) Tang, Q.; Harrata, A. K.; Lee, C. S. Anal. Chem. 1995, 67, 3515. (10) Tang, Q.; Harrata, A. K.; Lee, C. S. Anal. Chem. 1997, 69, 3177. (11) Jensen, P. K.; Pasa-Tolic, L.; Anderson, G. A.; Horner, J. A.; Lipton, M. S.; Bruce, J. E.; Smith, R. D. Anal. Chem. 1999, 71, 2076. (12) Wall, D. B.; Kachman, M. T.; Gong, S.; Hinderer, R.; Parus, S.; Misek, D. E.; Hanash, S. M.; Lubman, D. M. Anal. Chem. 2000, 72, 1099. (13) Kachman, M. T.; Wang, H.; Schwartz, D. R.; Cho, K. R.; Lubman, D. M. Anal. Chem. 2002, 74, 1779. (14) McCormack, A. L.; Schieltz, D. M.; Goode, B.; Yang, S.; Barnes, G.; Drubin, D.; Yates, J. R., III. Anal. Chem. 1997, 69, 767.

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phase separation techniques fully exploit the sensitivity achievable with conventional mass spectrometers (roughly 10-16 mol, as opposed to 10-14 mol in conjunction with 2-D PAGE), allowing many additional proteins to be identified.21 Furthermore, direct analysis of peptide mixtures obtained from the digestion of complex cell lysates offers much greater potential for the analysis of low-abundance proteins than does 2-D PAGE.16,24 Still, large quantities of cellular proteins or enzymatically/ chemically cleaved peptides ranging from a few milligrams13,14,24,25 to several hundred micrograms15-17,21-23 are typically needed to increase the proteome coverage, particularly for the identification of low-abundance proteins. Additionally, only limited sample amounts ranging from 103 to 105 cells are generally available in mammalian proteomics, corresponding to a total protein content of 0.1-10 µg. For example, the use of laser capture microdissection techniques yields tissue volumes in cubic micrometers and sample sizes in the submicrogram range.26 Furthermore, the heterogeneous nature of cells and tissues also contributes to the requirement for analyzing limited subpopulations. However, the sensitive and routine characterization of the entire spectrum of proteins, including low-abundance proteins, is essential to proteome-wide protein identification and quantitative expression profiling and still challenges the development of various bioanalytical technologies, from sample processing to separation and MS detection. For example, the reduction of chromatography column inner diameters from 350-180 µm21-25 to 100-15 µm15,20,27-30 results in higher analyte concentrations within smaller peak volumes, thus enabling more sensitive MS detection. Moreover, a dynamic range enhancement approach is applied to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) for increasing proteome coverage in quantitative peptide abundance measurements.31 It should be emphasized that the extremely high resolution of 2-D PAGE for protein separation is mostly contributed by (15) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nature Biotechnol. 1999, 17, 676. (16) Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nature Biotechnol. 2001, 19, 242. (17) Wolters, D. A.; Washburn, M. P.; Yates, J. R., III. Anal. Chem. 2001, 73, 5683. (18) Washburn, M. P.; Ulaszek, R.; Deciu, C.; Schieltz, D. M.; Yates, J. R., III. Anal. Chem. 2002, 74, 1650. (19) Li, W.; Hendrickson, C. L.; Emmett, M. R.; Marshall, A. G. Anal. Chem. 1999, 71, 4397. (20) Martin, S. E.; Shabanowitz, J.; Hunt, D. F.; Marto, J. A. Anal. Chem. 2000, 72, 4266. (21) Shen, Y.; Zhao, R.; Belov, M. E.; Conrads, T. P.; Anderson, G. A.; Tang, K.; Pasa-Tolic, L.; Veenstra, T. D.; Lipton, M. S.; Udseth, H. R.; Smith, R. D. Anal. Chem. 2001, 73, 1766. (22) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nature Biotechnol. 1999, 17, 994. (23) Han, D. K.; Eng, J.; Zhou, H.; Aebersold, R. Nature Biotechnol. 2001, 19, 946. (24) Gygi, S. P.; Rist, B.; Griffin, T. J.; Eng, J.; Aebersold, R. J. Proteome Res. 2002, 1, 47. (25) VerBerkmoes, N. C.; Bundy, J. L.; Hauser, L.; Asano, K. G.; Razumovskaya, J.; Larimer, F.; Hettich, R. L.; Stephenson, J. J., Jr. J. Proteome Res. 2002, 1, 239. (26) Bonner, R. F.; Emmert-Buck, M. R.; Cole, K.; Pohida, T.; Chuaqui, R.; Goldstein, S.; Liotta, L. A. Science 1997, 278, 1481. (27) Hsieh, S.; Jorgenson, J. W. Anal. Chem. 1996, 68, 1212. (28) MacNair, J. E.; Lewis, K. C.; Jorgenson, J. W. Anal. Chem. 1997, 69, 983. (29) Quenzer, T. L.; Emmett, M. R.; Hendrickson, C. L.; Kelly, P. H.; Marshall, A. G. Anal. Chem. 2001, 73, 1721. (30) Shen, Y.; Zhao, R.; Berger, S. J.; Anderson, G. A.; Rodriguez, N.; Smith, R. D. Anal. Chem. 2002, 74, 4235.

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isoelectric focusing in the first separation dimension. By transferring isoelectric focusing separation from gel to capillary format, the focusing effect of capillary isoelectric focusing (CIEF) not only contributes to a high-resolution protein/peptide separation with a pI difference as small as 0.005 pH unit,32,33 but also provides a typical concentration factor of at least 100 times. Thus, on-line combination of CIEF with capillary reversed-phase liquid chromatography (CRPLC) has been developed for performing 2-D analysis of Drosophila proteomics using only UV detection in our previous studies.34 Our research efforts in this work further expand, validate, and optimize the technical basis for effectively concentrating peptides/ proteins as the result of analyte focusing, followed by multidimensional separation of complex mixtures using combined CIEF with CRPLC. The grouping of two highly resolving techniques, which consist of completely orthogonal separation mechanisms, together with analyte focusing/concentration drastically enhances the dynamic range and sensitivity of conventional electrospray ionization (ESI) tandem MS toward the identification of lowabundance proteins. Our results clearly demonstrate the ability to identify a larger number of yeast proteins than those presented in the literature. Most significantly, all of these measurements are carried out with a protein loading which is 2-3 orders of magnitude lower than those employed by the current non-gelbased proteome techniques.13-17,21-25 The distribution of the codon adaptation index (CAI) value among identified proteins is similar to that predicted for the entire yeast proteome16 and further supports the great potential of CIEF-based multidimensional separation technology for the comprehensive and ultrasensitive analysis of complex proteomes. EXPERIMENTAL SECTION Materials and Reagents. Fused-silica capillaries (100 µm i.d./ 200 µm o.d. and 100 µm i.d./365 µm o.d.) were acquired from Polymicro Technologies (Phoenix, AZ). Ammonium hydroxide, acetic acid, dithiothreitol (DTT) were obtained from Sigma (St. Louis, MO). Acetonitrile, DNase, glycerol, hydroxypropyl cellulose (av MW 100 000), magnesium chloride, tris(hydroxymethyl)aminomethane (Tris), and urea were purchased from Fisher Scientific (Pittsburgh, PA). Heptafluorobutyric acid (HFBA) and Pharmalyte 3-10 were acquired from Pierce (Rockford, IL) and Amersham Pharmacia Biotech (Uppsala, Sweden), respectively. All solutions were prepared using water purified by a Nanopure II system (Dubuque, IA) and further filtered with a 0.22-µm membrane (Costar, Cambridge, MA). Tryptic Digest of Soluble Protein Extract from Saccharomyces cerevisiae. The yeast cells (Sigma) were suspended in a buffer which consisted of 10 mM Tris (pH 7.0), 5 mM magnesium chloride, 0.1 mM DTT, and 10% glycerol. The cells were disrupted by sonication for the release of cellular proteins.35 (31) Pasa-Tolic, L.; Harkewicz, R.; Anderson, G. A.; Tolic, N.; Shen, Y.; Zhao, R.; Thrall, B.; Masselon, C.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2002, 13, 954. (32) Conti, M.; Gelfi, C.; Righetti, P. G. Electrophoresis 1995, 16, 1485. (33) Conti, M.; Galassi, M.; Bossi, A.; Righetti, P. G. J. Chromatogr., A 1997, 757, 237. (34) Chen, J.; Baehrecke, E. H.; Shen, Y.; Smith, R. D.; Lee, C. S. Electrophoresis 2002, 23, 3143. (35) Schleif, R. F.; Wensink, P. C. Practical Methods in Molecular Biology; SpringerVerlag: New York, 1981; Chapter 1.

Figure 1. Schematic of on-line integration of CIEF with CRPLC as a concentrating and multidimensional separation platform. Solid and dashed lines represent the flow paths for the loading of CIEF fractions and the injection of fractions into a CRPLC column, respectively.

After sonication, DNase was added with a final concentration of 50 µg/mL for the cleavage and removal of nucleic acids. The cellular proteins were collected in the supernatant by centrifugation at 20000g for 10 min. The protein solution was then desalted using a regenerated cellulose membrane (Millipore, Bedford, MA) with a 5000 molecular weight cutoff. The total protein concentration was determined using the Bradford method (BioRad, Richmond, CA) and was ∼2 mg/mL. Yeast cytosol proteins were denatured and reduced in a 20 mM Tris buffer containing 8 M urea and 0.1 M DTT. Trypsin (1:50, w/w, Promega modified sequencing grade, Madison, WI) was added, and the mixture was incubated at 37 °C overnight. CIEF-Based Multidimensional Concentration/Separation Platform. A schematic overview for the on-line integration of CIEF with CRPLC as a concentrating and multidimensional separation platform is shown in Figure 1. A 60-cm CIEF capillary (100 µm i.d./200 µm o.d.) was coated with hydroxypropyl cellulose for the elimination of electroosmotic flow and protein/peptide adsorption onto the capillary wall.34 The capillary was initially filled with a solution containing 2% Pharmalyte 3-10 and 2 mg/mL tryptic peptides obtained from the soluble fraction of yeast cell lysates. There were no column performance changes for more than 20 runs of CIEF separations using the coated capillary. The stability of hydroxypropyl-coated capillaries was further supported by the work of Shen and co-workers.36 The solutions of 0.5% ammonium hydroxide at pH 10.5 and 0.1 M acetic acid at pH 2.5 were employed as the catholyte and the anolyte, respective. Focusing was performed at an electric field of 300 V/cm over the entire CIEF capillary. The current decreased continuously as the result of analyte focusing. Once the current reduced to ∼10% of the original value, usually within 30 min, the (36) Shen, Y.; Smith R. D. J. Microcolumn Sep. 2000, 12, 135.

focusing was considered to be complete. The focused peptides were sequentially and hydrodynamically loaded into a 0.4-µL injection loop in a 6-port microinjection valve (Upchurch Scientific, Oak Harbor, WA). The loaded peptides were then injected into a C18 reversed-phase trap column using a Harvard Apparatus 22 syringe pump (Holliston, MA) through a 6-port microselection valve (Upchurch Scientific). Repeated peptide loadings and injections into various trap columns were carried out until the entire CIEF capillary content was sampled into a total of 12 unique fractions. A constant electric field of 300 V/cm was applied across the CIEF capillary for maintaining analyte band-focusing in the capillary throughout the loading and injection procedures. Trap columns were prepared in-house. The effluent end of a 8-cm-long fused-silica capillary (100 µm i.d./365 µm o.d.) was connected to an inline microfilter assembly (Upchurch Scientific). The C18-bonded particles (5-µm diameter, 100-Å pores, Phenomenex, Torrance, CA) in methanol were introduced into the capillary by gradually increasing the pressure from 100 to 2000 psi using an Agilent capillary LC pump (Avondale, PA). The capillary packed with 3 cm of C18-bonded particles was left under pressure for 10 h and then depressurized overnight. Because of the highly charged and hydrophilic nature of carrier ampholytes (Pharmalyte 3-10) employed for the creation of a pH gradient during the CIEF step, these ampholytes were eluted from the trap columns toward a waste reservoir prior to the CRPLC separations using a 0.5% acetic acid solution. An Agilent capillary LC pump was then employed to generate a 90-min linear gradient from 5 to 65% acetonitrile (containing 0.02% HFBA) at a flow rate of 1 µL/min. Through the use of a second 6-port microselection valve (Upchurch Scientific), the mobile phase was delivered into the individual trap column, followed by a 15-cm-long capillary column (180 µm i.d. × 365 µm o.d., LC Packings, San Francisco, CA) packed with 5-µm porous C18 reversed-phase particles. All 12 isoelectric focusing fractions collected in the trap columns were analyzed in sequence from acidic to basic pIs, and the eluants from CRPLC were monitored using a ThermoFinnigan LCQ ion trap mass spectrometer (San Jose, CA). Multidimensional Concentration/Separation Platform Using Nano-RPLC. The tip at the end of a 16-cm-long fused-silica capillary (100 µm i.d. × 365 µm o.d.) was flame-pulled and packed with 12.5 cm of 5-µm C18-bonded particles (Phenomenex). This nano RPLC column was employed as the second separation dimension for further resolving isoelectric focusing fractions on the basis of their differences in hydrophobicity. A microcross (Upchurch Scientific) containing a platinum electrode was employed to apply an ESI voltage of 1.7 kV and reduce the flow of capillary LC pump from 2 µL/min to an effective flow rate of 0.2 µL/min. A linear gradient from 5 to 45% acetonitrile (containing 0.02% HFBA) was applied over 90 min, and the gradient was then increased from 45 to 80% in 20 min and stayed at 80% for another 10 min. Data Acquisition and Analysis. The LCQ was operated via Instrument Method files in the Sequence Setup window of Xcalibur. The heated desolvation capillary was set to 180 °C. For a 12-step 2-D separation experiment, each isoelectric focusing fraction was represented by one Instrument Method file with identical settings. In the Instrument Method files, the LCQ was set to acquire a full MS scan between 500 and 1700 m/z followed Analytical Chemistry, Vol. 75, No. 13, July 1, 2003

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Figure 2. Base peak chromatograms of a representative CIEF/CRPLC multidimensional separation of 9.6 µg of yeast tryptic peptides obtained from the soluble fraction of cell lysates. Each number represents the sequence of CIEF fractions further analyzed by CRPLC from acid to basic pHs.

by five MS/MS scans of the top five ions from the preceding full MS scan. Relative collision energy for collision-induced dissociation was set to 35% with a 30-ms activation time. Dynamic exclusion was enabled with a repeat count of 2, a repeat duration of 0.5 min, and a 10-min exclusion duration window. The SEQUEST algorithm was used to interpret MS/MS data.15,37 A peptide was considered to be a match by utilizing the following constraints: (i) a minimum cross-correlation score (Xcorr) of 1.9, 2.2, and 3.75 for singly, doubly, and triply charged peptides, respectively; (ii) a requirement that returned peptides had fully tryptic ends; and (iii) enforcement of a minimum ∆Cn score of at least 0.1. For proteins identified by three or more (37) Yates, J. R., III; Carmack, E.; Hays, L.; Link, A. J.; Eng, J. K. Methods Mol. Biol. 1999, 112, 553.

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qualifying and unique peptides, no manual assessment of spectra was conducted. For proteins being identified by fewer unique peptides possessing SEQUEST scores that passed the above criteria, at least one of the SEQUEST results was visually assessed using criteria described previously15 to confirm or deny the presence of a protein. RESULTS AND DISCUSSION An important aspect of any multidimensional separation platform is its ability to improve the detection of analytes present in low quantities during the analyses of complex protein/peptide mixtures. The use of CIEF as the first separation dimension provides both sample concentration and excellent resolving power in this study. For example, the typical analyte bandwidth after focusing has been observed around 2.5 mm inside a 60-cm-long

Figure 3. The range of pI values in each of the 12 CIEF fractions displayed in Figure 2.

CIEF capillary by applying an electric field strength of 300 V/cm over a pH gradient from 3 to 10. This yields to a sample concentration factor of ∼240 and a baseline resolution (resolution, Rs, of 1.5) of ∼160 peaks. In isoelectric focusing, the resolving power, ∆(pI)min, is controlled by

∆(pI)min ) 3 {(D/E) [(dpH/dx)/(-dµ/dpH)]}1/2 (1) where D is the diffusion coefficient of the species, E is the applied electric field strength, dpH/dx is the pH gradient, and dµ/dpH is the change of protein mobility against solution pH.38 The separation power can, therefore, be further enhanced by raising the applied voltage (and thus, the applied electric field strength) by assuming the absence of Joule heating. Maximum achievable resolution can be obtained through voltage programming by ramping up the voltage during the focusing step to avoid Joule heating, since the current decreases continuously as the result of protein focusing. The combination of various ampholytes can be utilized for the formation of both narrow and wide pH gradients for isoelectric focusing separation. In situations in which enhanced resolution of yeast peptides with similar pI values is desired, the use of narrow range ampholyte mixtures may be employed (see eq 1). Narrow-range ampholyte mixtures generating gradients spanning 1-3 pH units are available from many commercial sources. For the separation of yeast tryptic digest from the soluble fraction of cell lysates with broadly different pIs, a wide-range ampholyte, e.g., Pharmalyte 3-10, was employed in this study. However, very basic peptides with pIs g11 may not be resolved or could be lost to the catholyte using the currently available carrier ampholytes. Still, our recent studies have demonstrated the use of N,N,N′,N′tetramethylethylenediamine for extending the separation range to at least pI 12.39 CIEF-Based Multidimensional Concentration/Separation Platform. Figure 2 shows the base peak chromatograms of a (38) Rodriguez-Diaz, R.; Wehr, T.; Zhu, M.; Levi, V. Capillary Isoelectric Focusing. In Handbook of Capillary Electrophoresis; Landers, J. P., Ed.; CRC Press: Boca Raton, FL, 1997; p 101. (39) Mohan, D.; Lee, C. S. J. Chromatogr., A 2002, 979, 271.

representative CIEF/CRPLC analysis of yeast tryptic peptides obtained from the soluble fraction of cell lysates. The peptides in each isoelectric focusing fraction were further separated by CRPLC and identified using ESI-MS/MS. As reported previously by Wolters and co-workers,17 acetic acid or trifluoroacetic acid in the chromatography mobile phase was substituted by HFBA to increase the analyte concentrations in the eluted peaks with sharpened peak shape. In the integrated CIEF/CRPLC approach, peptides were systematically resolved by their differences in pI and hydrophobicity. This was evidenced by the correlation of the peptide pI value versus the CIEF fraction number (from acidic to basic pIs) shown in Figure 3. By using 2% Pharmalyte 3-10, we were able to identify the peptides over a wide pH range of at least 3.8-10.2. The pI range analyzed in this study was comparable to that reported using strong cation exchange chromatography.17 However, the degree of pI overlapping in CIEF fractions was drastically lower than that in strong cation exchange chromatography using a salt gradient at acidic pH. For example, the percentage of identified peptides present in more than one CIEF fraction was around 10-25% (data not shown) and significantly less than 40-80% obtained from multidimensional LC using strong cation exchange coupled with reversed-phase separations.17 Reproducibility of peptide separation and identification using the combined CIEF/CRPLC/ESI-MS/MS approach was examined by performing multiple runs of identical yeast tryptic digest samples. For two analyses performed on consecutive days, the elution times of the same peptides in CRPLC differed by 6000 proteins). Proteins from all functional classes are clearly not accessible by analyzing the soluble fraction only. A total of 1484 yeast proteins have therefore been identified by pooling the data from three fractions of cell lysates, including the soluble fraction, the lightly washed insoluble fraction, and the heavily washed insoluble fraction.16,17 By using only the soluble fraction, our studies already demonstrate the capabilities of CIEF-based multidimensional separation technology for identifying a larger number of soluble yeast proteins than other techniques presented in the literature. It should be emphasized that the competitive advantages of our multidimensional separation platform are mostly attributed to high 3150 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003

Figure 5. Distribution of CAI values for identified yeast proteins using the CIEF/CRPLC multidimensional separation platform.

resolving power and analyte concentration effect in CIEF. Most importantly, the amount of tryptic peptides employed for performing yeast proteome analysis was only ∼9.6 µg (2 mg/mL × 4.8 µL of capillary volume in CIEF) which is 2-3 orders of magnitude less than those utilized in the current non-gel-based proteome techniques.13-17,21-25 Instead of using significantly higher amounts

Figure 6. Mass spectrum of chromatography peak eluted at 32.21 min for the CRPLC separation of the first CIEF fraction (see Figure 4A). The subsequent tandem mass spectra identify the coelution of high- (YFL014W) and low- (YMR107W) abundance proteins.

of sample materials and additional sample fractionation procedures, the combination of analyte focusing/concentration with two highly resolving and orthogonal separation mechanisms in an integrated platform significantly enhances both the dynamic range and the sensitivity of conventional mass spectrometry toward the proteome analysis. It is thought that CAI is a measure of protein abundance, because highly expressed proteins generally have a CAI value.16 The yeast genes with a CAI