A Proteome Strategy for Fractionating Proteins and Peptides Using

Anal. Chem. 2004, 76, 4811-4824

A Proteome Strategy for Fractionating Proteins and Peptides Using Continuous Free-Flow Electrophoresis Coupled Off-Line to Reversed-Phase High-Performance Liquid Chromatography Robert L. Moritz,† Hong Ji,† Fre´de´ric Schu 1 tz,‡ Lisa M. Connolly,† Eugene A. Kapp,† ‡,§ Terence P. Speed, and Richard J. Simpson*,†

Joint ProteomicS Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch)/The Walter and Eliza Hall Institute of Medical Research, Parkville, 3050, Victoria, Australia, and Department of Statistics, University of California, Berkeley, California 94720

Extensive prefractionation is now considered to be a necessary prerequisite for the comprehensive analysis of complex proteomes where the dynamic range of protein abundances can vary from ∼106 for cells to ∼1010 for tissues such as blood. Here, we describe a high-resolution 2D protein separation system that uses a continuous freeflow electrophoresis (FFE) device to fractionate complex protein mixtures by solution-phase isoelectric focusing (IEF) into 96 well-defined pools, each separated by ∼0.02-0.10 pH unit depending on the gradient created, followed by rapid (∼6 min per analysis) reversed-phase high-performance liquid chromatography (RP-HPLC) of each FFE pool. Fractionated proteins are readily visualized in a virtual 2D format using software that plots protein loci, pI in the first dimension and relative hydrophobicity (i.e., RP-HPLC retention time) in the second dimension. By coupling a diode-array detector in line with a multiwavelength fluorescence detector, separated proteins can be monitored in the RP-HPLC eluent by both UV absorbance and intrinsic fluorescence simultaneously from a single experiment. Triplicate analyses of standard proteins using a pH 3-10 gradient conducted over a 3-day period revealed a high system reproducibility with a SD of 0.57 (0.05 pH unit) within the FFE pools and 0.003 (0.18 s) for protein retention times in the seconddimension RP-HPLC step. In addition, we demonstrate that the FFE-IEF/RP-HPLC separation strategy can also be applied to complex mixtures of low molecular weight compounds such as peptides. With the facile ability to measure the pH of the isoelectric focused pools, peptide pI values can be estimated and used to qualify peptide identifications made using either MS/MS sequencing approaches or pI discriminated peptide mass fingerprint* Corresponding author. Tel: +61 3 9341 3155. Fax: +61 3 9341 3192. E-mail: [email protected]. † Ludwig Institute for Cancer Research. ‡ The Walter and Eliza Hall Institute of Medical Research. § University of California. 10.1021/ac049717l CCC: $27.50 Published on Web 07/09/2004

© 2004 American Chemical Society

ing. The calculated peak capacity of this 2D liquid-based FFE-IEF/RP-HPLC system is 6720. A number of key technical challenges need to be overcome before proteomics can realize its full potential for protein expression profiling of biological matrixes such as cells and tissues.1,2 Foremost is the problem of dynamic range of protein abundances. For instance, the dynamic range of protein abundances in a cell can be as high as 106, with low-abundance proteins such as transcription factors present at ∼10 copies/cell and the moreabundant cell structure proteins at ∼1 000 000 copies/cell. This makes it extremely challenging to visualize low-abundance proteins in complex proteomes, let alone identify them using current mass spectrometry (MS)-based identification methods.3-5 Of equal importance is the problem that proteins exhibit tremendous heterogeneity with respect to size, charge, posttranslational modifications, and solubility. Consequently, a wide range of protein separation methods, or combinations thereof (i.e., multidimensional separation strategies), are usually required for the comprehensive analysis of complex proteomes. For several decades, two-dimensional gel electrophoresis (2DE)6,7 has been the only proteomics technique that has permitted the separation of thousands of proteins in a single experiment.8,9 Recently, the resolving power of 2-DE has been extended significantly with the introduction of narrow-range immobilized pH gradients and extended separation distances.10,11 Additionally, improvements in choice of detergent12-15 and detergent extraction (1) Simpson, R. J. Proteins and Proteomics: A Laboratory Manual; Cold Spring Harbor Laboratory Press: New York, 2003. (2) Simpson, R. J. Purifying proteins for proteomics: A laboratory Manual; Cold Spring Harbor Laboratory Press: New York, 2004. (3) Domon, B.; Alving, K.; He, T.; Ryan, T. E.; Patterson, S. D. Curr. Opin. Mol. Ther. 2002, 4, 577-86. (4) Patterson, S. D. Biotechniques 2003, 35, 440-4. (5) Ferguson, P. L.; Smith, R. D. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 399-424. (6) O’Farrell, P. H. J. Biol. Chem. 1975, 250, 4007-21. (7) Klose, J. Humangenetik 1975, 26, 231-43. (8) Go ¨rg, A.; Obermaier, C.; Boguth, G.; Harder, A.; Scheibe, B.; Wildgruber, R.; Weiss, W. Electrophoresis 2000, 21, 1037-53. (9) Lottspeich, F. Angew. Chem., Int. Ed. 1999, 38, 2477-92.

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strategies16,17 have significantly increased the number of proteins (especially, membrane-associated proteins) that can be visualized using 2-DE. Nevertheless, the dynamic range of protein abundances that can be separated by 2-DE is ∼104, which is too low to cover that encountered in many cell types and tissues such as blood (∼109-1010). Several approaches for overcoming the protein dynamic range impasse have been recently described. These include the following: (1) the disassembly of the macromolecular architecture of cells into their constituent organelles, macromolecular structures, and multiprotein complexes; proteome analysis can then be performed on subcellular components of interestsi.e., subproteome analysis;18,19 (2) enrichment of cellular components using differential detergent fractionation20,21 and molecular weight fractionation;22 (3) depletion of abundant proteins using nonbiospecific chromatographyse.g., dye ligands such as Cibacron-Blue and derivatives thereof for the removal of serum albumin,23 immunoaffinity chromatography using specific antibodies against abundant proteins, and removal of IgG from serum using immobilized protein-A or -G columns;24-28 (4) selective precipitation, for example, with trifluoracetic acid/acetone29 prior to 2-DE; (5) various multidimensional chromatography methods.30,31 Prefractionation of proteins from large volumes, as a prelude to subsequent analysis using 2-DE, can be performed by preparative polyacrylamide gel electrophoresis (PAGE) on the basis of apparent molecular mass (Mr), usually in the presence of ionic detergents (e.g., SDS) and a preparative SDS-PAGE apparatus such as the Bio-Rad model 491 PrepCell.32,33 Alternative electrophoretic prefractionation methods based upon solution-phase (10) Hochstrasser, D. F.; Sanchez, J. C.; Appel, R. D. Proteomics 2002, 2, 80712. (11) Go ¨rg, A.; Postel, W.; Gunther, S. Electrophoresis 1988, 9, 531-46. (12) Rabilloud, T.; Blisnick, T.; Heller, M.; Luche, S.; Aebersold, R.; Lunardi, J.; Braun-Breton, C. Electrophoresis 1999, 20, 3603-10. (13) Rabilloud, T.; Strub, J. M.; Luche, S.; van Dorsselaer, A.; Lunardi, J. Proteomics 2001, 1, 699-704. (14) Luche, S.; Santoni, V.; Rabilloud, T. Proteomics 2003, 3, 249-53. (15) Tastet, C.; Charmont, S.; Chevallet, M.; Luche, S.; Rabilloud, T. Proteomics 2003, 3, 111-21. (16) Bordier, C. J. Biol. Chem. 1981, 256, 1604-7. (17) Pryde, J. G.; Phillips, J. H. Biochem. J. 1986, 233, 525-33. (18) Jung, E.; Heller, M.; Sanchez, J. C.; Hochstrasser, D. F. Electrophoresis 2000, 21, 3369-77. (19) Brunet, S.; Thibault, P.; Gagnon, E.; Kearney, P.; Bergeron, J. J.; Desjardins, M. Trends Cell Biol. 2003, 13, 629-38. (20) Ramsby, M. L.; Makowski, G. S.; Khairallah, E. A. Electrophoresis 1994, 15, 265-77. (21) Ramsby, M. L.; Makowski, G. S. Methods Mol. Biol. 1999, 112, 53-66. (22) Ji, H.; Baldwin, G. S.; Burgess, A. W.; Moritz, R. L.; Ward, L. D.; Simpson, R. J. J. Biol. Chem. 1993, 268, 13396-405. (23) Travis, J.; Pannell, R. Clin. Chim. Acta 1973, 49, 49-52. (24) Bjorck, L.; Kronvall, G. Acta Pathol. Microbiol. Scand. [B] 1981, 89, 1-6. (25) Bjorck, L.; Kronvall, G. J. Immunol. 1984, 133, 969-74. (26) Akerstrom, B.; Brodin, T.; Reis, K.; Bjorck, L. J. Immunol. 1985, 135, 258992. (27) Guss, B.; Eliasson, M.; Olsson, A.; Uhlen, M.; Frej, A. K.; Jornvall, H.; Flock, J. I.; Lindberg, M. EMBO J. 1986, 5, 1567-75. (28) Govorukhina, N. I.; Keizer-Gunnink, A.; van der Zee, A. G. J.; de Jong, S.; de Bruijn, H. W. A.; Bischoff, R. J. Chromatogr., A 2003, 1009, 171-8. (29) Go ¨rg, A.; Boguth, G.; Obermaier, C.; Weiss, W. Electrophoresis 1998, 19, 1516-9. (30) Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-7. (31) Wu, C. C.; MacCoss, M. J. Curr. Opin. Mol. Ther. 2002, 4, 242-50. (32) Zugaro, L. M.; Reid, G. E.; Ji, H.; Eddes, J. S.; Murphy, A. C.; Burgess, A. W.; Simpson, R. J. Electrophoresis 1998, 19, 867-76. (33) Fountoulakis, M.; Juranville, J. F. Anal. Biochem. 2003, 313, 267-82.

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isoelectric focusing (IEF), rather than gel-based IEF34 used in 2-DE, have been developed. These non-gel-based IEF methods can accommodate large volumes and, in contrast to gel-based IEF methods, are not limited by sample amount. Preparative IEF as a prefractionation technique was first proposed by Bier’s laboratory35,36 and Rotafor, a commercial version of this apparatus, was produced by Bio-Rad. Because this apparatus has no separation barriers, low resolution and large volumes are typically encountered. A modified form of this apparatus with improved performance has recently been described by Shang and colleagues.37 To reduce sample complexity and enrich for low-abundance proteins, the Rotofor has been used to fractionate large volumes of biological fluids, in combination with SDS-PAGE gels or matrix-assisted laser desorption/ionization time-of-flight analysis)38,39 or reversed-phase high-performance liquid chromatography (RP-HPLC).40 To overcome problems associated with the original Rotofor device, Righetti and colleagues41 developed a multicompartment electrolyzer (IsoPrime, Amersham) where each compartment was separated by a polyacrylamide gel membrane (embedded with covalently attached immobilines analogous to those used in IPG gels), each with a defined pH. Further improvements to this apparatus have been made by rotating the chamber instead of using a peristaltic pump and immersing the chamber in a cooling bath.42 A variation of this device, where there are five separation disks installed, creating a maximum of seven pH separation chambers (42-mL total volume, 6 mL/chamber), has been commercialized (MCE-IsoelectricQ, Proteome Systems). A microscale liquid-phase IEF prefractionation method that is also based upon a multicompartment apparatus (4.7-mL total volume, 650 µL/chamber) has recently been described by Zuo and Speicher.43 In this apparatus, the chambers are separated by thin polyacrylamide gels containing ampholyte mixtures at specific pH values; this device is now commercially available (Zoom IEF Fractionator, Invitrogen). Another liquid-based IEF prefractionation technology, the Gradiflow system,44 comprises recirculating hydraulic flow of the protein mixtures through two shallow separation compartments with an orthogonal electrophoretic transport of different proteins across a single separation membrane between the recirculating compartments. Unlike most other multicompartment electrokinetic analyzers, the Gradiflow technology allows the electrophoretic separation of proteins based upon both a protein’s charge and molecular shape (size) properties.45 (34) Bjellqvist, B.; Ek, K.; Righetti, P. G.; Gianazza, E.; Go¨rg, A.; Westermeier, R.; Postel, W. J. Biochem. Biophys. Methods 1982, 6, 317-39. (35) Egen, N. B.; Bliss, M.; Mayersohn, M.; Owens, S. M.; Arnold, L.; Bier, M. Anal. Biochem. 1988, 172, 488-94. (36) Bier, M.; Long, T. J. Chromatogr. 1992, 604, 73-83. (37) Shang, T. Q.; Ginter, J. M.; Johnston, M. V.; Larsen, B. S.; McEwen, C. N. Electrophoresis 2003, 24, 2359-68. (38) Davidsson, P.; Westman, A.; Puchades, M.; Nilsson, C. L.; Blennow, K. Anal. Chem. 1999, 71, 642-7. (39) Nilsson, C. L.; Puchades, M.; Westman, A.; Blennow, K.; Davidsson, P. Electrophoresis 1999, 20, 860-5. (40) 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, 1099-111. (41) Faupel, M.; Barzaghi, B.; Gelfi, C.; Righetti, P. G. J. Biochem. Biophys. Methods 1987, 15, 147-61. (42) Herbert, B.; Righetti, P. G. Electrophoresis 2000, 21, 3639-48. (43) Zuo, X.; Speicher, D. W. Proteomics. 2002, 2, 58-68. (44) Ogle, D.; Sheehan, M.; Rumbel, B.; Gibson, T.; Rylatt, D. B. J. Chromatogr., A 2003, 989, 65-72. (45) Rylatt, D. B.; Napoli, M.; Ogle, D.; Gilbert, A.; Lim, S.; Nair, C. H. J. Chromatogr., A 1999, 865, 145-53.

For a recent review of prefractionation techniques in proteome analysis, see Righetti et al.46 and Simpson.2 Another liquid-based IEF prefractionation technique, continuous free-flow electrophoresis (FFE), first described by Hannig,47,48 continuously injects samples into a carrier ampholine solution flowing as a thin laminar film (0.3-1.0 mm wide) between two plates. By introducing an electric field perpendicular to the direction of flow, proteins (as well as lower molecular weight species) can be separated by IEF according to their different pI values and subsequently collected for proteome analysis.49,50 Previously, we reported an uncoupled FFE-IEF/SDS-PAGE strategy for separating cytosolic proteins from a human colon carcinoma cell line for subsequent identification by on-line RPHPLC/electrospray ionization (ESI)-ion trap (IT) MS.50 In this present study, we have further refined this strategy by introducing off-line rapid RP-HPLC (1-6 min separation times) as a second dimension for each of the FFE fractions. By using a judicious choice of ampholytes in the first-dimension FFE step, we demonstrate that IEF can be performed over both broad and narrow ranges of pH. Additionally, we have developed software to present the chromatographic output as a single 2D plot (virtual 2D analysis) for quick evaluation and “spot” matching of fractionated proteins. By coupling a diode-array detector (UV detection from 190 to 400 nm) in-line with a multiwavelength fluorescence detector (using multiple excitation and emission wavelengths) extra levels of information (or multiplexing) can be mined from a single experiment. The efficacy of this strategy is demonstrated using a standard set of proteins, the total cellular lysate of the colon carcinoma cell line LIM 1215,51 a tryptic digest of this total cellular lysate, and a human urine specimen. EXPERIMENTAL SECTION Materials. Hydroxypropyl methyl cellulose (HPMC) (viscosity, 2 wt % in H2O, 4000 cP; nominal Mr, 86K) and iodoacetic acid (IAA) was obtained from Aldrich (Milwaukee, WI). Carrier ampholytes for preparative IEF were purchased from Serva (Servalytes) (Heidelberg, Germany) or from Sinus (Sinulytes) (Heidelberg, Germany). Protein standards: chick egg ovalbumin pI 4.7 Mr 45K; BSA pI 4.9 Mr 67K; bovine carbonic anhydrase pI 6.18 Mr 30K; horse myoglobin pI 6.88, 7.33 Mr 17.5K; bovine ribonuclease b pI 8.88, Mr 13.5K; bovine cytochrome c pI 9.28 Mr 12.2K; chick egg lysozyme pI 9.3, Mr 14.3K; and human insulin pI 5.4, Mr 5.5K were from Sigma (St. Louis, MO). The 96 standardand deep-well rack systems and pressure-sensitive plate closures were from Greiner (Frickenhausen, Germany). PD-10 and FastDesalting columns were from Amersham-Biosciences. Sequencing-grade trypsin (EC 3.4.21.4) was from Worthington. Trifluoroacetic acid (TFA, HPLC/Spectro grade) and tris(2carboxyethyl)phosphine (TCEP) were from Pierce (Rockford, IL). Acetonitrile and methanol (ChromAR grade) were purchased from Mallinckrodt (Melbourne, Australia), and glacial acetic acid (46) Righetti, P. G.; Castagna, A.; Herbert, B.; Reymond, F.; Rossier, J. S. Proteomics. 2003, 3, 1397-407. (47) Hannig, K. Fresenius Z. Anal. Chem. 1961, 181, 244-74. (48) Hannig, K.; Heidrich, H. G. Free-Flow Electrophoresis; GIT Verlag GmbH: Darmstadt, 1990. (49) Burggraf, D.; Weber, G.; Lottspeich, F. Electrophoresis 1995, 16, 1010-5. (50) Hoffmann, P.; Ji, H.; Moritz, R. L.; Connolly, L. M.; Frecklington, D. F.; Layton, M. J.; Eddes, J. S.; Simpson, R. J. Proteomics 2001, 1, 807-18. (51) Simpson, R. J.; Connolly, L. M.; Eddes, J. S.; Pereira, J. J.; Moritz, R. L.; Reid, G. E. Electrophoresis 2000, 21, 1707-32.

(UnivAR grade) was from BDH (Merck, Australia). Unless otherwise stated, all chemicals used were of analytical grade. Deionized water, obtained from an A10-Synthesis water polishing system (Millipore) was used for all solutions. LIM 1215 Colon Carcinoma Cell Culture. Human colon carcinoma cell line LIM 121552 was routinely passaged in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum, R-thioglycerol (10-5 M), insulin (25 units/L), and hydrocortisone (1 mg/L), as previously described.51 After LIM 1215 cells reached 70% confluency, the cells were scraped off the tissue culture dishes (150-mm diameter), washed three times with PBS, and harvested by centrifugation at 480g. Resultant cell pellets were used for cellular protein fractionation. Cellular Protein Fractionation and Tryptic Digestion. Cell pellets (∼2 × 108 cells) were resuspended in 15 mL of ice-cold cytosolic buffer (10 mM HEPES, 120 mM KCl, 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO3, 1 mM CaCl2, 0.5 mM MgCl2, pH 7.1)53 containing Complete, EDTA-free protease inhibitor cocktail tablets (1 tablet/25 mL). The cell suspension was placed in a cell disruption bomb (PARR Instrument Co.), pressurized to 700 psi for 15 min, and cell membranes were disrupted by the sudden release of nitrogen pressure as they were forced out of the thin tubular orifice. The disrupted cell suspension was then centrifuged at 900g for 10 min to remove nuclei fraction. The supernatant containing membrane and cytosolic proteins was centrifuged at 25000g for 20 min to remove membrane proteins, and the resultant supernatant was collected (cytosolic fraction). The salt content of the cytosolic fraction was reduced by passing the fraction through a PD-10 column equilibrated with 5 mM Tris-HCl, pH 8.0. For FFE analysis, the protein concentration of the cytosol fraction was adjusted to 2 mg/mL proteins by dilution with a solution of 0.4% (w/v) HPMC containing 0.8% (v/v) ampholytes followed by water to achieve a final concentration of 0.2% (w/v) HPMC and 0.4% (v/v) ampholytes. For tryptic digestion of the LIM 1215 cytosol proteins, 5 mg (1 mL) of the sample was adjusted to 4.8 M with GnHCl and then reduced with TCEP to a final concentration of 10 mM for 30 min at 70 °C. Following reduction, IAA was added to the solution to a final concentration of 100 mM and the solution incubated for 30 min in the dark. Following alkylation, the solution was buffer exchanged with 1% ammonium bicarbonate using a Fast-desalting column operated at 1 mL/min at ambient temperature. Trypsin was added to a final ratio of 1:20 and the mixture incubated for 16 h at 37 °C. Following digestion, the tryptic digest was lyophilized and then reconstituted in 25% (v/v) glycerol, 0.4% (v/ v) ampholytes for FFE-IEF. Fractionation of Urinary Proteins. Human urine (500 mL) from a healthy male individual was first treated with the addition of 1 M NaCL and incubated at 4 °C overnight in order to precipitate Tamm-Horsfall protein (uromodulin).54 Following precipitation, the supernatant was carefully drawn off, centrifuged at 2000g to pellet any remaining protein, and then dialyzed extensively (3 days at 4 °C) against water using 2000 MWCO (52) Whitehead, R. H.; Macrae, F. A.; St John, D. J.; Ma, J. J. Natl. Cancer Inst. 1985, 74, 759-65. (53) Muldoon, L. L.; Jamieson, G. A., Jr.; Villereal, M. L. J. Cell Physiol. 1987, 130, 29-36. (54) Grover, P. K.; Marshall, V. R.; Ryall, R. L. Clin. Sci. (London) 1994, 87, 137-42.


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Figure 1. Schematic representation of the continuous FFE apparatus coupled off-line to RP-HPLC. Dimensions of focusing chamber are 50 × 10 × 0.4 cm (10-cm distance between electrodes, 45-cm electrode length). For analytical imaging separations, a portion of each first-dimension FFE-IEF fraction (50 µL/total volume, ∼2 mL) was injected directly from the 96-deep-well plate using the Agilent 1100 HPLC equipped with a well-plate autosampler.

dialysis tubing (Spectra/Por 6 RC, Spectrum Laboratories). The dialyzed urine was freeze-dried and then dissolved in FFE buffer (0.2% w/v HPMC, containing 0.4% v/v ampholytes) to a final protein concentration of 2 mg/mL. Electrophoresis Procedures. (1) Preparative SolutionPhase IEF Using Continuous Free-Flow Electrophoresis. FFE (Figure 1) was performed using the Octopus apparatus (Dr. Weber GmbH (Kirchheim, Germany) described elsewhere.50 The IEF separation medium was either aqueous 0.2% (w/v) HPMC or 25% v/v glycerol containing 0.4% (v/v) carrier ampholytes (Servalyte pH 3-10, 4-6, 5-7, 9-11, or Sinulyte pH 2-4, 3-5, 7-9, 2-11). The electrode solutions were 100 mM H3PO4 (anode) and 50 mM NaOH (cathode). The counterflow (0.7 mL/min) was either aqueous 0.2% (w/v) HPMC or 25% (v/v) glycerol. IEF separation conditions and stabilization times for the apparatus were optimized by allowing the separation buffer to achieve a stable current of ∼17 mA. The electrophorectic separation was monitored using a mixture of colored proteins (aqueous 0.5 mg/mL bovine cytochrome c (pI 9.10), horse myoglobin (pI 6.85 and 7.05), BSA (pI 4.80), and 0.001% bromophenol blue, diluted 1:1 with separation medium). The pH gradient formed was determined by measuring the pH of each of the 96 fractions collected with a combination glass microtipped pH electrode (model B10C161, Radiometer) connected to a standard pH meter (model PHM210, Radiometer). For preparative FFE separations, samples were adjusted 1:1 with a solution containing 0.4% (w/v) HPMC and 0.8% (v/v) carrier ampholytes and then further diluted with running buffer (0.2% (w/v) HPMC containing 0.4% (v/v) carrier ampholytes) to a final concentration of 1-2.5 mg of protein/mL. Samples were introduced into the FFE apparatus through one of the main sample ports (S1 (acidic), S2 (neutral), or S3 (basic)) at a flow rate of 1.4-1.8 mL/min. IEF was performed at 4 °C with a constant voltage of 1250 V (∼12-17 mA). The sample residence time in the separation chamber was 20 min. After stabilizing the IEF 4814


separation for 20 min, fractions (∼2 mL) were collected in a deep 96-well rack. Between runs, the apparatus was washed extensively with water. Fractions were stored at 4 °C for subsequent RP-HPLC or SDS-PAGE, or at -20 °C for long-term storage. (2) Two-Dimensional Electrophoresis. 2-DE was performed on protein mixtures under nonreducing conditions as previously described.55 First-dimensional IEF was performed on an IPGphor electrophoresis apparatus using 24-cm Immobiline Drystrips, pH 3-10. Protein samples were applied to Immobiline Drystrips using the rehydration method described in the manufacturer’s instructions. The IEF focusing conditions were 500 V for 1 h, 1000 V for 1 h, 1000-8000 V gradient for 1 h, and 8000 V for 13 h. After first-dimensional focusing, the Immobiline strips were equilibrated 2 × 15 min with 20 mL of reducing SDS sample buffer (50 mM Tris-HCl, pH 7.5, 2% (w/v) SDS, 75 mM DTT). The seconddimension SDS-PAGE was performed on a Multiphor II electrophoresis unit utilizing Excelgel (8-18% gradient acrylamide) precast gels. Proteins were visualized by FAST Coomassie Brilliant Blue staining (Fisher Biotec). Rapid Reversed-Phase High-Performance Liquid Chromatography. RP-HPLC was performed using an 1100 HPLC system (Agilent Technologies) equipped with a well plate autosampler and column compartment heater. Column eluent was monitored using a diode-array detector fitted with a standard 13µL flow cell (Agilent model G1315B) and a multiwavelength fluorescent detector fitted with a standard 8-µL flow cell (Agilent model G1321A) coupled in-line. FFE sample fractions (50 µL) were injected directly onto a Brownlee RP-300 cartridge (100 mm × 2.1 mm i.d., octylsilica 300-Å pore size, 7-µm dp (Perkin-Elmer)). The column was developed at 1 mL/min over 6 min at 40 °C using a linear 6-min gradient from 0 to 100% B, where solvent A was (55) Ji, H.; Moritz, R. L.; Reid, G. E.; Ritter, G.; Catimel, B.; Nice, E.; Heath, J. K.; White, S. J.; Welt, S.; Old, L. J.; Burgess, A. W.; Simpson, R. J. Electrophoresis 1997, 18, 614-21.

0.1% TFA in water and solvent B was 0.094% TFA, 60% CH3CN, water. Column eluent was monitored for both UV absorption at 200, 215, 254, 280, and 290 nm, and intrinsic protein fluorescence (both at 278-nm excitation (Ex)/360-nm emission (Em) and 295nm Ex/360-nm Em). For subsequent MS analysis, proteins fractions were collected manually after correcting for the postcolumn dead volume (50 µL). Two-Dimensional (2D) FFE-IEF/RP-HPLC Protein Profile Analysis by Virtual 2D Image Interpolation. Absorbance plots for all FFE fractions were extracted from their respective second-dimension RP-HPLC chromatograms by a macro running under Chemstation software (Agilent Technologies) to create a single comma-separated variable (CSV) file which contained both X and Y coordinates for each chromatogram as separate columns. Single CSV files were then imported and processed as interpolated image plots using Transform (Research Systems Inc.). Reproducibility of 2D FFE-IEF/RP-HPLC System. Three separate 2D FFE-IEF/RP-HPLC analyses were performed over three consecutive days to provide triplicate data to determine dayto-day reproducibility of the system. The nine most intense peaks located between wells 13 and 87 (the range of wells over which pH increases linearly), corresponding to nine different protein spots, were extracted from the RP-HPLC chromatograms, and the standard deviations (SDs) for both retention time and well numbers were calculated across the three replicates for each peak. The results were pooled and SDs calculated for both well number (FFE-IEF analysis) and retention time (RP-HPLC). To improve the reproducibility, the pH measurements of each individual well were used to adjust the well numbers across the different replicates. The measured pH value of each well was used to produce two different reference curves. The first reference curve was the average pH at each well number across the three experiments, with intermediate values calculated by linear interpolation. The second was the straight line fitted by least squares through all pH measures between wells 13 and 87 for the three replicates. These reference curves were then inverted to find the respective adjusted well number corresponding to the pH value of a given well. This ensured that if a peak was in three different wells across the replicates and the wells have the same pH, the adjusted well number would be the same for the three peaks. Protein Concentration Determination. Protein concentration of samples was determined using the bicinchoninic acid (BCA) procedure56 using BSA of known concentration to obtain a standard calibration curve. Protein Identification by Tryptic Digest and Mass Spectrometry. Protein fractions isolated from the second-dimension RP-HPLC step were digested manually with trypsin (0.05 µg) as described elsewhere.57 Tryptic digests were dried by centrifugal lyophilization (Savant model AES1010) and then redissolved in 10 µL of 0.1% aqueous TFA for subsequent identification by MS. MS-based peptide sequencing was performed using an ESI-IT mass spectrometer (model LCQ or LCQ-DECA; Thermo-Finnigan, San Jose, CA) coupled on line with a standard HPLC system (Hewlett-Packard model 1090A) that had been modified for (56) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985, 150, 76-85. (57) Moritz, R. L.; Eddes, J. S.; Reid, G. E.; Simpson, R. J. Electrophoresis 1996, 17, 907-17.

capillary chromatography51,57 or an 1100 capillary HPLC (Agilent). Operating conditions for ESI-IT-MS and MS data analysis are described elsewhere.51 Automatically selected tryptic peptide ions were identified using the SEQUEST algorithm (version 27, rev 12) incorporated into the Finnigan Xcalibur software (version 1.3)58 or the MASCOT algorithm (version 1.9.05, Matrix Science).59 A nonredundant protein database (created by searching through and then removing all redundant protein sequences in 13 FASTA formatted databases; SwissProt, SwissProt_variant_splices, TrEMBL, TrEMBL_new, TrEMBL_variant_splices (produced by the Swiss Institute of Bioinformatics and the European Bioinformatics Institute); GenBank, genpep, genpep_updates, yeastpep, wormpep (produced by NCBI); pir1, pir2, pir3, and pir4 (produced by PIR) was produced by the Office of Information Technology of the Ludwig Institute for Cancer Research and obtained from the Swiss Institute of Bioinformatics by public FTP (URL: ftp://ftp.ch.embnet.org/pub/databases/nr_prot/) on a weekly basis, which is then concatenated and sorted so that all human entries followed by seven other closely related species are brought to the top of the list) is stored as a single FASTA formatted database for use with MS/MS search algorithms. This formatted database, currently comprising 1 411 806 entries (594 Mb), was used for all searches. As well as minimizing any redundancies, this database also includes variant splice isoforms not currently found in other nonredundant databases. Simplified data reduction was performed using the CHOMPER data reduction program.60 Edman Degradation. A biphasic-column Edman sequencer (Agilent Technologies, model G1009A) was used for N-terminal peptide sequencing, as described elsewhere.57 RESULTS AND DISCUSSION Previously, we described a non-2-DE proteome analysis strategy using FFE coupled off-line to SDS-PAGE.50 However, the use of SDS-PAGE in the second dimension presents a serious disadvantage because of its restricted separation capacity, with only a limited number of proteins being separated, as well as the additional limitations such as recovery of low-Mr proteins and sample loadability. For these reasons, we have evaluated RP-HPLC as an alternative second-dimension separation step to be used after liquid-phase FFE-IEF purification where a single sample is separated initially into 96 fractions. Rapid RP-HPLC. To provide a facile second-dimension separation system that could be completed within a working day, we explored the use of rapid RP-HPLC on conventional wide-pore silica columns61 as a separation mode orthogonal to liquid-phase IEF. A separation of eight standard proteins using a variety of different linear flow velocities, but not modifying the gradient volume, is shown in Figure 2. In panel A, 1 µg each of the standard proteins ranging from Mr ∼5.5K to 67K and pI values ∼4.7-9.3 is chromatographed at a superficial linear velocity of 1730 cm/h (1 mL/min) with a gradient time of 6 min (i.e., 100 µL/% CH3CN change). Inspection of the chromatogram shown in Figure 2A (58) Eng, J. K.; Mccormack, A. L.; Yates, J. R. J. Am. Soc. Mass Spectrom. 1994, 5, 976-89. (59) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551-67. (60) Eddes, J. S.; Kapp, E. A.; Frecklington, D. F.; Connolly, L. M.; Layton, M. J.; Moritz, R. L.; Simpson, R. J. Proteomics. 2002, 2, 1097-103. (61) Moritz, R. L.; Eddes, J.; Simpson, R. J. Methods in Protein Sequence Analysis; Atassi, A., Appela, E., Eds.; Plenum Press: New York, 1995.


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Figure 2. High-speed RP-HPLC separation of standard proteins. Standard proteins loaded: bovine ribonuclease b (pI 8.88, Mr 13.5K); bovine cytochrome c (pI 9.28, Mr 12.2K); human insulin (pI 5.4, Mr 5.5K); chick egg lysozyme (pI 9.3, Mr 14.3K); BSA (pI 4.9, Mr 67K); bovine carbonic anhydrase (pI 6.18, Mr 30K); horse myoglobin (pI 6.88, 7.33 Mr 17.5K); chick egg ovalbumin (pI 4.7, Mr 45K). Chromatographic conditions: column, Brownlee RP-300, 7-µm dp, 300-Å C8 100 mm × 2.1 mm cartridge. Buffer A, 0.1% TFA in water; buffer B, 0.094% TFA/60% CH3CN/water; temperature, 40 °C. (Panel A) Gradient: 0-100% B in 6 min (1.0 mL/min, superficial linear flow velocity of 1732 cm/h). (Panel B) Gradient: 0-100% B in 60 min (0.1 mL/min, superficial linear flow velocity of 173 cm/h).

reveals that the resolution is maintained at this high flow rate when compared to the same set of proteins chromatographed on the same column, but at a lower linear velocity of 173 cm/h (0.1 mL/min) and with a gradient time of 60 min but identical gradient volume (Figure 2B). Virtual 2D Imaging of FFE-IEF/RP-HPLC Data. Easily interpretable data representations are paramount in assessing the quality of multiplexed separation strategies. To this end, we have developed software routines to extract the data from multiple chromatographic files and combined them into a standard format for importing into software graphing packages. In Figure 3, we compare the resolution of a standard set of proteins resolved using conventional nonreducing 2-DE (Figure 3A) and our 2D FFE-IEF/ RP-HPLC system (Figure 3B). The apparent pI values of proteins contained in individual FFE fractions are readily determined by measuring the pH value for each FFE fraction using a microtipped combination pH electrode. These data are plotted on the righthand axis of the 2D FFE-IEF/RP-HPLC profile, the pH gradient being superimposed on the 2D plot. Once these data are imported into Transform, both low- and high- abundance proteins can be readily visualized by adjusting the optical density (OD) scale. A comparison of Figure 3B and C reveals that by changing the OD from 2.0 AUFS to 0.01 AUFS, low-abundance contaminants from the commercial protein standards used in the study can be visualized. In Figure 4, those protein standards displayed topographically from the merged set of RP-HPLC chromatograms were plotted as a 3D image in order to ascertain whether they were individual 4816 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004

proteins and not two or more colocalizing protein peaks (for example, the eight protein peaks from the FFE-IEF/RP-HPLC data set are essentially homogeneous). This can be accomplished without having to refer to individual RP-HPLC chromatograms. Reproducibility of 2D FFE-IEF/RP-HPLC Separation Strategy. To assess the day-to-day reproducibility of the FFEIEF/RP-HPLC separation system, a representative triplicate set of data was obtained for seven of the protein standards over three consecutive days. The proteins used to calculate the positional change in both pI and retention time were chosen to include the widest range in pI (i.e., pI 4.7-9.3), a broad range in molecular weight (i.e., ∼12K-67K) as well as apparent surface hydrophobicity. As shown, the triplicate analyses obtained over the three-day period (Figure 5A-C) is highly reproducible in both the FFEIEF and RP-HPLC loci for each protein. When the measured pH gradients for the triplicate experiments are overlaid (Figure 5D), excellent concordance is observed over the entire pH range of the gradient. To further analyze the reproducibility, SD calculations were performed over the three sets of data (Figure 5E). The pooled SD for the retention time across three replicates for each of the nine peaks (seven major protein peaks with the addition of two minor peaks from the impure protein preparations) was 0.003, indicating a high degree of reproducibility on this axis. By contrast, the pooled SD for uncorrected FFE well number was 0.83. When the FFE well number was adjusted, using the three sample reference pH curves and an average of these, the standard deviation was reduced to 0.62. With regression analysis through pH measurements between wells 13 and 87, the data showed good

Figure 3. Separation of protein standards by nonreducing 2-DE and 2D FFE/RP-HPLC. (Panel A) Nonreducing 2-DE of standard proteins: bovine ribonuclease b (pI 8.88, Mr 13.5K); bovine cytochrome c (pI 9.28, Mr 12.2K); chick egg lysozyme (pI 9.3, Mr 14.3K); BSA (pI 4.9, Mr 67K); bovine carbonic anhydrase (pI 6.18, Mr 30K); horse myoglobin (pI 6.88, 7.33 Mr 17.5K); chick egg ovalbumin (pI 4.7, Mr 45K). Sample: 20 µg of each protein was loaded and the resultant gel stained with Fast Coomassie Blue. (Panel B) Native two-dimension FFE-IEF (pH 3-10)/RPHPLC separation of standard proteins. Sample, standard proteins loaded were identical to those described in panel A; sample load, 5-mL protein mixture (0.25 mg each protein/mL of FFE primary buffer). FFE conditions: aqueous 0.2% (w/v) HPMC containing 0.4% (v/v) Servalyte pH 3-10. IEF was conducted using the electrophoretic conditions described in the Experimental Section. Proteins were detected in the seconddimension RP-HPLC at 215 nm at 2.0 AU. (Panel C) High-sensitivity profile of native two-dimension FFE-IEF (pH 3-10)/RP-HPLC separation of standard proteins shown in panel B, but plotted at 0.01 AU.

Figure 4. Three-dimensional visualization of a 2D FFE/RP-HPLC separation of standard proteins. Native two-dimension FFE-IEF (pH gradient 3-10)/RP-HPLC separation of standard proteins as shown in Figure 3 plotted as a three-dimensional representation. Peak intensity (z axis) absorbance plot at 215 nm.

correlation (R2 ) 0.993), and then using this regression data as a reference, further reduced the SD to 0.57, indicating good reproducibility over consecutive days of operation. Taken together, these values compare favorably to current 2D-gel-based systems of immobilized pH gradient gels in the first dimension and SDS gels in the second dimension where SD values of 0.3-0.5 ( 0.3% can be achieved.62 Narrow-Range pH Separations Using FFE. To further increase the resolution of the IEF mode in the first-dimension (62) Lopez, M. F.; Patton, W. F. Electrophoresis 1997, 18, 338-43.

FFE step, narrow-range pH gradients of 1-2 units can be readily formed over both acidic (Figure 6A-D) and basic pH ranges (Figure 6E and F) by judicious use of carrier ampholytes. These simple pH gradients can be formed to overlap by a single pH unit or, alternatively, adjacent to each other to optimize protein separation. The difference in pH between the FFE fractions created by a broad-range pH gradient (e.g., pH 2-11; Figure 6H) is in the order of 0.08 pH unit, whereas narrow-range pH gradients (e.g., pH 4-6; Figure 6C) can produce differences as little as 0.02 pH unit. Unlike standard gel- and static volume-based IEF Analytical Chemistry, Vol. 76, No. 16, August 15, 2004

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Figure 5. Reproducibilty of FFE pH gradient formation. Individual native two-dimension FFE-IEF (Servalyte pH 3-10)/RP-HPLC separation of standard proteins as shown in Figure 3 performed over three consecutive days. (Panels A-C) FFE-IEF/RP-HPLC of seven standard proteins (as shown in Figure 3) electrophoresed by FFE-IEF at pH 3-10 in the first dimension and then chromatographed by RP-HPLC in the second dimension (conditions as described in Figure 3). (Panel D) Overlay of pH gradient reproducibility created by FFE over three consecutive days and linear regression analysis of the three data sets. (Panel E) Pooled standard deviation of FFE/RP-HPLC separation system (for calculation of SD values, see Experimental Section).

approaches34,35,41-43 where protein accumulation may occur at the edges of the pH gradient, in laminar flow-based FFE-IEF separations, this potential problem is minimized due to the continual flushing of the separated sample. Multiplex Proteomic Analysis Using UV Absorption and Intrinsic Protein Fluorescence. With the development of onthe-fly UV diode-array detection and multiple-wavelength fluorescence detection, multiple-wavelength analysis as well as real-time spectral information can be collected simultaneously for peaks eluting from RP-HPLC.63 Here we have applied this technology in order to increase the information content of the 2D FFE-IEF/ RP-HPLC separation profiles. As shown in Figure 7, multiplewavelength plots for both UV (Figure 7, panels A and B) and fluorescence detection (Figure 7, panels C and D) can easily be obtained on single samples. Of the protein standards used, only ribonuclease is devoid of tryptophan but contains both phenylalanine and tyrosine, which can be used to detect proteins by UV absorbance at wavelengths (e.g., 254-290 nm) higher than 214 nm, which is used to detect peptide bonds. It can be seen in Figure 7D that the loci for ribonuclease is not present when fluorescence is used to specifically detect tryptophan residues at an excitation of 295 nm and emission of 360 nm but present if the wavelengths of Ex 280 nm and Em 360 nm are used (Figure 7C). The direct coupling of the column effluent to a multiple-wavelength fluorescence detection system provides a facile technique in which to probe the amino acid composition of proteins during fractionation. (63) Grego, B.; Nice, E. C.; Simpson, R. J. J. Chromatogr. 1986, 352, 359-68.

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Peak Capacity for 2D FFE-IEF/RP-HPLC Protein Separations. The qualifying parameter for any multidimensional separation technique is peak capacity.64 The accepted number of proteins resolved in a well-optimized single RP-HPLC step is in the order of ∼100-130 peaks.65,66 The low-flow rate RP-HPLC step shown here (Figure 2B) has a calculated peak capacity of 117. Given that the linear range of the first-dimension broad pH gradient (∆pH ∼0.1) FFE-IEF step presented here consists of ∼80 fractions, where proteins are fractionated into single wells, we have calculated the peak capacity of our 2D FFE-IEF/RP-HPLC system utilizing low-flow rate RP-HPLC to be ∼9360. By incorporating rapid RP-HPLC in our strategy, the peak capacity for this step is reduced by ∼28% due to diffusion effects of the stationary phase employed, thereby giving an overall 2D FFE-IEF/rapid RP-HPLC system peak capacity of ∼6720. This value is significantly higher than the peak capacity value of 2640 recently reported for a micropreparative system based on capillary IEF (peak capacity, 12) and capillary RP-HPLC (peak capacity, 220).67 Although the peak capacity number assumes that multiple proteins or peptides would be present at all pH values of the FFE-IEF step and (64) Apffel, A. Purifying proteins for proteomics: A Laboratory Manual; Simpson, R. J., Ed.; Cold Spring Harbor Laboratory Press: New York, 2004; Chapter 4. (65) Cheng, Y.; Lu, Z.; Neue, U. Rapid Commun. Mass Spectrom. 2001, 15, 14151. (66) Wagner, K.; Miliotis, T.; Marko-Varga, G.; Bischoff, R.; Unger, K. K. Anal. Chem. 2002, 74, 809-20. (67) Chen, J.; Balgley, B. M.; DeVoe, D. L.; Lee, C. S. Anal. Chem. 2003, 75, 3145-52.

Figure 6. Narrow-range pH separation of protein standards using FFE/RP-HPLC. Native 2D FFE-IEF/RP-HPLC separation of standard proteins performed at 1250 V and 11-17 mA. Electrophoretic buffer: aqueous 0.2% (w/v) HPMC, containing the following: (panel A) 0.4% (v/v) Sinulyte (Sinus GmbH, Germany) pH 2-4, (panel B) Servalyte (SERVA GmbH, Germany) pH 3-5, (panel C) Sinulyte (Sinus GmbH, Germany) pH 4-6, (panel D) Sinulyte (Sinus GmbH, Germany) pH 5-7, (panel E) Servalyte (SERVA GmbH, Germany) pH 7-9, (panel F) Sinulyte (Sinus GmbH, Germany) pH 9-11, (panel G) Servalyte (SERVA GmbH, Germany) pH 3-10, and (panel H) Sinulyte (Sinus GmbH, Germany) pH 2-11. Counterflow buffer: aqueous 0.2% (w/v) HPMC. Standard protein mixture (see legend to Figure 3 for protein standard details) of 0.5 mg each protein/mL loaded.

Figure 7. Multiplexing of in-line diode-array and fluorescence detection for protein standards using a single FFE-IEF/RP-HPLC analysis. 2D liquid FFE-IEF/RP-HPLC separation of standard proteins as indicated in Figure 3B. (Panel A) 215 nm; (panel B) 280 nm; (panel C) fluorescence excitation 280 nm, emission 360 nm; (panel D) excitation 295 nm, emission 360 nm.

retention times during the RP-HPLC separation stages, in practice this is often not observed.64 It is generally accepted that the working peak capacity of the RP-HPLC step can be much lower due to the unpredictable nature of proteins extracted from whole cells (e.g., 10-20% of the theoretical capacity) therefore reducing the overall system peak capacity accordingly. By introducing a third orthogonal dimension into the separation system (e.g., size or accurate mass analysis), the peak capacity of the system can be significantly increased. For example, using a scan range spanning 1600 m/z units (scan range of 400-2000 m/z) operated at unit mass resolution and with the operation of

dynamic exclusion during the tandem-MS step, where peptide ions are excluded for a period of 2 min after initial selection for fragmentation (i.e., effective m/z scan range divided by the exclusion window),68 results in a practical peak capacity of ∼213 600 (80 (FFE fractions) × 10 (RP-HPLC reduced peak capacity) × 267 (m/z range of 1600 divided by dynamic exclusion window of 6 mass units)). Given these conservative estimates, the working peak capacity of the liquid-based FFE-IEF coupled offline to RP-HPLC and subsequently tandem-MS system is still (68) Cargile, B. J.; Bundy, J. L.; Freeman, T. W.; Stephenson, J. L. J. Proteome Res. 2004, 3, 112-9.


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of sufficient capacity for the analysis of complex proteomes such as human tissue samples. Our approach to separate whole proteins with FFE-IEF as a first dimension, using only low-molarity buffer components, ampholytes, and low concentrations of HPMC or glycerol as a viscosity modifier, capable of nondenaturing and nonreducing separations, results in high pI discrimination of as low as 0.02 pH unit. Because there are no physical barriers for proteins or lowMr compounds to transgress or bind to, this enables the purification of both small and very large Mr compounds, in their native state, as well as their respective protein-protein complexes (unpublished results). In our experience, the FFE device is robust and the operation and maintenance of the device is relatively simple; over 7 years of operation, only chamber seals, electrode filters, and sample and buffer tubing required replacement. This is in contrast to other lower resolution IEF based separation techniques where pH compartment separators35,41-43 or expensive microcolumn-based ion-exchange columns40 often need replacement due to sample contamination in the first dimension. Protein Recovery from 2D-FFE-IEF/RP-HPLC. The recovery of total cellular extract proteins from the colon carcinoma cell line LIM 1215 after FFE-IEF, as judged by BCA total protein assay performed in duplicate, was 87.6%. The recoveries of standard proteins after 2D-FFE-IEF/RP-HPLC, as judged by calculating the absorbance of the recovered individual protein HPLC peaks after fractionation as compared to an unfractionated starting mixture run under the same RP-HPLC conditions, was an average of 78.5% (data not shown). 2D-FFE-IEF/RP-HPLC Separation of Complex Tryptic Peptide Mixtures. It is well recognized that extensive fractionation of protein digests increases data density (i.e., the number of peptides in complex samples that can be identified by MS).69,70 In particular, peptides of low-abundance or low-ionization efficiency can be readily identified due to the minimization of suppression of ionization by co-present peptides by prior fractionation.71-74 Shotgun proteomics31 utilizes two dimensions in liquid chromatography, namely, ion exchange utilizing a stepped salt gradient and RP-HPLC, that are coupled in series and directly interfaced with tandem mass spectrometry. This approach permits the separation of abundant tryptic peptides from complex proteomes and their direct introduction into tandem mass spectrometers for subsequent identification.75,76 Here we have exploited the high resolution afforded by FFE-IEF/RP-HPLC to fractionate a total cellular lysate from the human colonic cell line LIM 1215 as well as a tryptic digest preparation of this extract (see Figure 8A and B). The separation capabilities of the 2D FFE-IEF/RP-HPLC system for cellular protein extracts is evident and is complemen(69) Liu, H. B.; Lin, D. Y.; Yates, J. R. Biotechniques 2002, 32, 898. (70) Washburn, M. P.; Ulaszek, R. R.; Yates, J. R. Anal. Chem. 2003, 75, 505461. (71) Kratzer, R.; Eckerskorn, C.; Karas, M.; Lottspeich, F. Electrophoresis 1998, 19, 1910-9. (72) Reid, G. E.; Rasmussen, R. K.; Dorow, D. S.; Simpson, R. J. Electrophoresis 1998, 19, 946-55. (73) Cech, N. B.; Enke, C. G. Anal. Chem. 2000, 72, 2717-23. (74) Burkitt, W. I.; Giannakopulos, A. E.; Sideridou, F.; Bashir, S.; Derrick, P. J. Aust. J. Chem. 2003, 56, 369-77. (75) Wolters, D. A.; Washburn, M. P.; Yates, J. R., III. Anal. Chem. 2001, 73, 5683-90. (76) Tabb, D. L.; McDonald, W. H.; Yates, J. R., III. J. Proteome Res. 2002, 1, 21-6.

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tary to many 2DE-gel-based systems. In Figure 8C, a single RPHPLC chromatogram corresponding to FFE well 38 is shown to illustrate the abundance of peptides isolated in a single pH fraction. Peptides fractionated in this manner can be readily analyzed by MS for accurate mass determination or sequenced by MS/MS techniques (Figure 8D). To date, our preliminary analysis has identified in excess of 254 peptides (77 individual proteins) using the 2D FFE-IEF/RPHPLC method, encompassing intracellular proteins that vary over a Mr range 11-119K. The length of tryptic peptides analyzed ranged from 6 to 36 residues and the theoretical peptide pI values varied from 3.66 to 12.00. For this preliminary study, only 0.8% of each selected FFE fraction was subjected to MS analysis. A comparison of theoretical and experimental pI values for a subset of identified peptides is given in Figure 9. It should be noted that the theoretical pI values for some peptides greatly exceeded the experimentally determined pH of the FFE well where they electrophoresed (e.g., peptide 183 AVFPSIVGRPR in well 82 has a theoretical pI value of 12.00 but was found present at a pH of 9.08). In some cases, this is due to the margin buffers compressing the end of the gradient formed by the focused ampholytes and therefore rapidly increasing or decreasing the pH of the cathode or anode margin buffer, respectively. For basic peptides, this problem can be overcome, partially, by separating the peptides over a narrow pH range that spans this pI value, as demonstrated for basic proteins (see Figure 6). Interestingly, a number of identified tryptic peptides with aberrant pI values were shown to be either N-terminally modified (e.g., N-terminally acetylated) or contain oxidized methionine (e.g., methionine sulfoxide), methylated histidine, or carboxymethyl cysteine (see Table 1). Use of pI as a Discriminator of MS-Based Peptide Identification. Because peptides can be separated by FFE-IEF into discrete narrow-range pH pools with defined pH values (see Figures 8B and 9), their pI values can be taken into consideration when validating MS-based peptide identification. FFE-IEF is an easier technique for estimating apparent pI values for peptides because the samples are in a liquid medium. This is a more facile approach than extracting peptides from frozen IPG gel strips in the method described by Cargile and co-workers.68 In a previous theoretical study, Cargile and Stephenson77 demonstrated that peptides of identical amino acid composition (isobaric mass) can have differing isoelectric points. It would be an attractive option, therefore, to incorporate a peptide’s pI value in a database search algorithm in order to increase the specificity of protein identification either by accurate-mass peptide mass fingerprinting (PMF) or MS/MS means. As well as increasing the confidence of peptide identification, the peptide search database size could be markedly decreased, resulting in a significant decrease in the overall search time. For every peptide mass with a tolerance value defined by the mass accuracy of the instrument used, the peptide database size reduction is accomplished by eliminating those peptides whose theoretical pI values are not in accordance with experimentally estimated pI values. The determination of a peptide’s theoretical pI value, compared to that of a full-length protein, is relatively trivial to compute because the likelihood of unknown posttranslational modifications (77) Cargile, B. J.; Stephenson, J. L. Anal. Chem. 2004, 76, 267-75.

Figure 8. Separation of human LIM 1215 cytosolic proteins and an unfractionated LIM 1215 cytosolic lysate tryptic digest by 2D FFE-IEF/ RP-HPLC. (Panel A) Sample: 1 mg of human LIM 1215 cytosolic proteins dissolved in 1 mL of IEF running buffer containing 0.2% (w/v) HPMC, 0.4% (v/v) Servalyte pH 3-10 loaded at 1.4 mL/h at 1500 V, 15 mA and collected into a deep 96-well plate. Second-dimension RP-HPLC separation of FFE-IEF fraction using conditions identical to those described in Figure 3B. (Panel B) Sample: 1 mg of human LIM 1215 cytosolic proteins digested with trypsin (1:20 enzyme/substrate ratio) dissolved in 1 mL of IEF running buffer containing 25% (v/v) glycerol, 0.4% (v/v) Servalyte pH 3-10 loaded at 1.4 mL/h at 1500 V, 15 mA and collected into a deep 96-well plate. Second-dimension RP-HPLC separation of FFE-IEF fraction using conditions identical to those described in panel A. (Panel C) Second-dimension RP-HPLC chromatogram for FFE fraction 38 (pH 5.56) from panel B (see fraction indicated in panel B). (Panel D) Tandem MS/MS spectrum of m/z 1159.2 [M + 2H]2+ (peptide selected for MS/MS as indicated by arrow in panel C) identified as peptide sequence IIPAIATTTAAVVGLVC*LELYK from human ubiquitin-activating enzyme E1 (SwissProt accession P22314 UBA1_HUMAN, see Supporting Information Table 1) (cysteine residue identified as carboxymethyl cysteine).

in peptides (see Table 1)78 is far less than those encountered in intact proteins. Currently, posttranslational modifications are ignored in all available algorithms for predicting pI values.79-81 Notwithstanding caveats such as posttranslational modifications and peptides that focus at the extreme of the pH gradient, it can be seen that the computed pI is in good agreement with the observed position of the experimentally determined pH of the FFE wells (Figure 9). In this preliminary study, the calculated pI values for most peptides was in close agreement with their experimentally determined pH focused position ((0.86 pH unit, SD of 0.50), indicating the usefulness of this approach as an additional independent criterion to support correct peptide identifications. In a similar vein, Smith and co-workers82 have proposed the use of a peptide’s elution time from an RP-HPLC column in combination with high-accuracy mass measurements for protein (78) Bjellqvist, B.; Hughes, G. J.; Pasquali, C.; Paquet, N.; Ravier, F.; Sanchez, J. C.; Frutiger, S.; Hochstrasser, D. Electrophoresis 1993, 14, 1023-31. (79) Skoog, B.; Wichman, A. Trac-Trends Anal. Chem. 1986, 5, 82-3. (80) Clauser, K. R.; Baker, P.; Burlingame, A. L. Anal. Chem. 1999, 71, 2871-82. (81) Gasteiger, E.; Gattiker, A.; Hoogland, C.; Ivanyi, I.; Appel, R. D.; Bairoch, A. Nucleic Acids Res. 2003, 31, 3784-8. (82) Strittmatter, E. F.; Ferguson, P. L.; Tang, K.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2003, 14, 980-91.

identification using discriminated PMF. This approach, while suitable for very small proteomes (e.g., Deinococcus radiodurans containing 3116 predicted open reading frames), is currently not feasible for larger mammalian proteomes. Furthermore, the use of a peptide’s elution time (hydrophobicity) is highly variable since even subtle variations in stationary or mobile phase, temperature, and flow rate does cause large variations in elution time. Hence, using RP-HPLC column selectivity for general protein identification in this manner is limited. Therefore, we believe the use of pI as a second discriminator for peptide identification by PMF or MS/ MS to be more widely applicable because FFE-IEF allows peptide pI value estimates in a facile manner. 2D-FFE-IEF/RP-HPLC Separation of Human Urinary Proteins. To demonstrate the biological efficacy of the FFE-IEF/ RP-HPLC strategy, we have applied the method to a preliminary proteomic analysis of urinary proteins. Because the TammHorsfall mucoprotein83,84 constitutes >40% all urinary proteins,85 this protein was first depleted from the urine sample by the addi(83) Tamm, I.; Horsfall, F. L. Proc. Soc. Exp. Biol. Med. 1950, 74, 108-14. (84) Tamm, I.; Horsfall, F. L. J. Exp. Med. 1952, 95, 71-97. (85) Fletcher, A. P. Glycoproteins: Their Composition, Structure and Function; Gottshalk, A., Ed.; Elsevier: New York, 1972.


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Figure 9. Comparison of theoretical and experimental pI values for tryptic peptides from LIM 1215 cytosolic lysate separated by 2D FFE-IEF (pH 3-10)/RP-HPLC. Box plots were automatically generated using the statistical package R, version 1.5.0 (http://www.r-project.org) using the default parameters (i.e., the box represents scores between 25 and 75%, with the median at 50%, outliners are scores >1.5 times the interquartile range (75-25%) from the box and are indicated by dots (O), whiskers (+ - +) extend to the highest or lowest score not considered to be an outlier). Table 1. Representative 2D FFE-IEF/RP-HPLC Fractionated Tryptic Peptides with Aberrant pI Values identified peptidesa

well no.

well pHb

pIc

∆pH

amino acid residues in sequence

Ac-DDDIAALVVDNGSG(MetOx)(cm-C)K Ac-EEEIAALVIDNGSG(MetOx)(cm-C)K Ac-EEEIAALVIDNGSGM(cm-C)K Ac-ADQLTEEQIAEFK S(cm-C)N(cm-C)LLLK Ac-ATAEVLNIGK

21 21 21 24 32 34

3.41 3.41 3.41 3.70 4.88 5.14

3.66 3.83 3.83 4.00 4.21 6.05

+0.25 +0.42 +0.42 +0.30 -0.67 +0.91

2-18 2-18 2-18 1-13 335-342 2-11

YPIE(me-H)GIVTNWDDMEK VIHDNFGIVEGL(MetOx)TTVHAITATQK

38 52

5.56 6.50

4.31 5.99

-1.25 -0.51

69-84 162-185

protein identificationd β-actin (P02570) γ-actin (P02571) γ-actin (P02571) calmodulin (P02593) R-enolaseP06733 purine pathway multifunctional protein (P22234) β-actin (P02570) glyceraldehyde 3-phosphate dehydrogenase (P04406)

a Ac, acetylated N-terminus, (me-H) methylated histidine, (MetOx) oxidized methionine, and (cm-C) carboxymethyl cysteine. b Manually measured using a microcombination laboratory pH electrode. c pI calculated using Protein Prospector.80 d SwissProt accession numbers are shown in parentheses.

tion of sodium chloride to 1 M concentration.54,84 Mucoprotein-depleted human urine was subjected to both nonreducing 2-DE (Figure 10A) and the 2D FFE-IEF/RP-HPLC system (Figure 10B) for comparison. Faint protein spots shown in FFE fraction 38 (pH 5.26) at an OD level of 0.15 AUFS can be readily visualized by referring back to the original RP-HPLC chromatogram corresponding to this FFE fraction (Figure 10C). In a separate RP-HPLC analysis of FFE fraction 38, protein peaks were collected manually for protein identification purposes (alternatively, protein peaks can be collected in a nonmanual mode using the Agilent low-volume fraction collection system (model G1387A). Protein peaks 1 and 2 from Figure 10C were identified by Edman degradation of the intact proteins, as well as by MS/MS analysis of derived tryptic peptides (data not shown), as human CD59 membrane protein (SwissProt entry CD59_HUMAN86) and human spasmolytic pep(86) Davies, A.; Simmons, D. L.; Hale, G.; Harrison, R. A.; Tighe, H.; Lachmann, P. J.; Waldmann, H. J. Exp. Med. 1989, 170, 637-54.

4822 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004

tide (TFF2) (SwissProt entry SP_HUMAN87), respectively. CD59, a 9 kDa polypeptide with a calculated pI of 5.2, has previously been identified as a gpi-anchored glycoprotein, with both N- and O-glycosylation. CD59 functions as an inhibitor of the complement membrane attack complex thereby preventing complement-mediated cell lysis88 and potentially limiting the efficacy of anticancer antibody therapy.89 TFF2 is a member of the trefoil family of small polypeptides (7-12 kDa, pI 5.2) that is secreted preferentially by gastric mucous neck cells and is thought to stimulate gastric mucosal healing by the inhibition of gastric acid secretion in gastrointestinal abnormalities including gastric and duodenal ulceration, Crohn disease, and Helicobacter pylori infection.90 To our knowledge, TTF2 has not been identified in human urine to date; (87) Tomasetto, C.; Rio, M. C.; Gautier, C.; Wolf, C.; Hareuveni, M.; Chambon, P.; Lathe, R. EMBO J. 1990, 9, 407-14. (88) Turnberg, D.; Botto, M. Mol. Immunol. 2003, 40, 145-53. (89) Fishelson, Z.; Donin, N.; Zell, S.; Schultz, S.; Kirschfink, M. Mol. Immunol. 2003, 40, 109-23.

Figure 10. Comparison of human urinary protein separations by nonreducing 2-DE and FFE-IEF/RP-HPLC. Sample: 1.25 mg of human urinary proteins dissolved in 5 mL of IEF running buffer containing 0.2% (w/v) HPMC, 0.4% (v/v) Servalyte pH 3-10. The sample was applied to the FFE apparatus through the central sample introduction port (S2, see Figure 1) at 1.4 mL/h. IEF was performed at 1250 V, 15 mA for 4 h, and eluent fractions were collected into a deep 96-well plate. Second-dimension RP-HPLC separations of individual FFE-IEF fractions (100 µL) were performed using conditions identical to those described in Figure 3B. (Panel A) nonreducing 2-DE; (panel B) 2D FFE-IEF/RP-HPLC (experimental conditions were identical to those used in Figure 3); (panel C) RP-HPLC chromatogram of FFE fraction 43 (pH 5.26) from panel B (see circled peaks). Peaks 1 and 2 were collected manually after correcting for postdetector dead volume, subjected to N-terminal Edman degradation. Peak 1 was identified as CD59 gpi-anchored membrane protein (LQXYNXPNP..., SwissProt-CD59_HUMAN) and peak 2 as spasmolytic polypeptide (EKPSPXQXSRL....., SwissProt-SP_HUMAN), respectively.

however, studies in rats where iodinated porcine TTF2 was administered by injection into the inferior vena cava and measured in various organs of the rat over a 24-h period, showed that 40% of the 125I-TTF2 was taken up by the kidney and excreted in the urine unmetabolized.91 The preparative 2D liquid-based separation system described here is capable of extending the dynamic range of protein detection due to the unlimited loadability in the primary FFEIEF step. For example, large quantities of protein or peptide can be fractionated initially for further orthogonal downstream fractionation. The ability to fractionate low-Mr compounds such as CD59 and TTF2, is an important feature of this 2D liquid-based FFE-IEF/RP-HPLC method because techniques designed for this purpose are underrepresented in the armory of current proteomic separation tools. For example, the effective resolution achieved by conventional 2D-gel electrophoresis is typically above 10 kDa. 2DE-gel-based systems as well as multicompartment and columnbased separation systems (for a review, see ref 46) are also limited by the quantity of bulk starting material that can be loaded in the first dimension (IPG). This limitation in fractionation of starting material can hamper efforts to mine complex tissues. For example, the dynamic range of plasma is purported to be ∼1010.92 The most abundant protein in human plasma is serum albumin (HSA) present at 40-50 mg/mL, whereas some of the least abundant proteins are cytokines such as interleukin-6 (IL-6)93 which is present at expected normal levels of ∼10 pg/mL. Given the (90) Farrell, J. J.; Taupin, D.; Koh, T. J.; Chen, D.; Zhao, C. M.; Podolsky, D. K.; Wang, T. C. J. Clin. Invest. 2002, 109, 193-204. (91) Poulsen, S. S.; Thulesen, J.; Nexo, E.; Thim, L. Gut 1998, 43, 240-7. (92) Anderson, N. L.; Polanski, M.; Pieper, R.; Gatlin, T.; Tirumalai, R. S.; Conrads, T. P.; Veenstra, T. D.; Adkins, J. N.; Pounds, J. G.; Fagan, R.; Lobley, A. Mol. Cell Proteomics 2004, 3, 311-26. (93) Simpson, R. J.; Hammacher, A.; Smith, D. K.; Matthews, J. M.; Ward, L. D. Protein Sci. 1997, 6, 929-55.

current sensitivity of routine protein and peptide identification by MS/MS is ∼500 amol, to obtain 500 amol of IL-6, ∼1.5 mL of plasma would be required for the initial fractionation step. However, this amount of plasma would also contain 1.4 µM (90 mg) HSA, which is 6.2 × 109 (w/w) in excess of IL-6. This is a formidable quantity of protein to fractionate and presents a challenge to current purification schemes where extensive prefractionatation/depletion strategies need to be invoked in order to reveal low-abundance proteins. However, this also assumes that the protein of interest is homogeneous and a 100% recovery is obtained through all fractionation steps. For IL-6, which is extensively posttranslationally modified (pI range 5-7, Mr range 22-29K93-95), much larger quantities of plasma would be required to obtain a single enriched population of IL-6 molecules for identification purposes. Other purification systems utilizing limitedvolume sample applications (e.g., Rotofor, Zoom IEF, etc.) or microsized dimensions (e.g., capillary IEF coupled to capillary RPHPLC utilizing low-nanoliter sample volumes)67 are not capable of fractionating sufficient starting material to mine these lowabundant proteins. The 2D FFE-IEF/RP-HPLC system described here, with its unlimited sample loadability in the first dimension is well suited for this purpose. Interestingly, the emerging “topdown” sequencing,96-102 technique for fully characterizing intact (94) Van Snick, J.; Cayphas, S.; Vink, A.; Uyttenhove, C.; Coulie, P. G.; Rubira, M. R.; Simpson, R. J. Proc. Natl. Acad. Sci. U.S.A 1986, 83, 9679-83. (95) Van Snick, J.; Vink, A.; Cayphas, S.; Uyttenhove, C. J. Exp. Med. 1987, 165, 641-9. (96) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-6. (97) Horn, D. M.; Zubarev, R. A.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10313-7. (98) Schaaff, T. G.; Cargile, B. J.; Stephenson, J. L., Jr.; McLuckey, S. A. Anal. Chem. 2000, 72, 899-907. (99) Shi, S. D.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty, F. W. Anal. Chem. 2001, 73, 19-22.


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proteins, including their posttranslational modifications, in highresolution mass spectrometers requires appropriate sample introduction methodologies; the 2D FFE-IEF/RP-HPLC system described here is ideally suited for this purpose. CONCLUSIONS A preparative 2D liquid-based protein and peptide fractionation strategy utilizing FFE (operated in an IEF mode) and coupled off-line to RP-HPLC is described. The advantages of this system are as follows: (i) protein or peptide separations in the first dimension (IEF) are performed in a liquid phase and, unlike other multicompartment electroanalyzers, are not restricted by passage through any barrier or matrix; (ii) the system is truly preparative by not being sample limited, and separation efficiency is maintained by continual flushing of the separated sample; (iii) the FFEIEF/RP-HPLC system is capable of separating compounds of both low-Mr (e.g., peptides) and high-Mr compounds (e.g., native proteins and their multimeric complexes) over a broad pH range. For those proteins (especially membrane proteins) that exhibit poor solubility at or near their pI value, an appropriate buffer (e.g., amino acids as well as detergents in the case of membrane protein separations) can be incorporated in the FFE counterflow media prior to collection in the 96-well plate to minimize the time that such proteins stand at their pI value.50 The high resolving power produced in the first-dimension IEF step, where very narrow range pH gradients can easily be generated, coupled to the high resolution of modern RP-HPLC stationary phases, extends the resolving power of this 2D protein separation system over other previously described 2D systems based solely on coupled HPLC columns.103,104 In the case of high-Mr proteins and very hydrophobic proteins such as membrane proteins, the RP-HPLC stationary phases can be substituted with other chromatographic modes such as hydrophobic interaction chromatography or hydroxyapatite stationary phases to extend the power of the method to cover classes of proteins that are refractory to RP chromatography. Additionally, we demonstrate that complex mixtures of tryptic peptides can be fractionated by 2D liquid-based FFE-IEF/RPHPLC. Although peptide, and subsequently protein, identification was performed using tandem-MS methods,51 it should be noted that FFE-IEF separation enables peptides to be fractionated into discrete pools with increasing pH values that vary one from another by ∼0.02 pH unit. The ease and rapid determination of the apparent peptide pI value can be achieved by measuring the pH of these pools using a laboratory combination pH electrode. (100) Sze, S. K.; Ge, Y.; Oh, H.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A 2002, 99, 1774-9. (101) Meng, F.; Cargile, B. J.; Patrie, S. M.; Johnson, J. R.; McLoughlin, S. M.; Kelleher, N. L. Anal. Chem. 2002, 74, 2923-9. (102) Nemeth-Cawley, J. F.; Tangarone, B. S.; Rouse, J. C. J. Proteome Res. 2003, 2, 495-505. (103) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 161-7. (104) Wagner, K.; Racaityte, K.; Unger, K. K.; Miliotis, T.; Edholm, L. E.; Bischoff, R.; Marko-Varga, G. J. Chromatogr., A 2000, 893, 293-305.

4824


By applying a peptide’s pI determinant to high mass accuracy (e1 ppm) PMF,105-107 the discrimination of peptides differing by small mass units or even isobaric peptides is feasible.77 However, to utilize the apparent pI of isolated peptides in a matching algorithm, further work needs to be performed on pI prediction algorithms to take into account posttranslational modifications, either biological such as acetylation, phosphorylation, etc., or chemical as a result of sample preparation/processing (e.g., methionine oxidation, S-alkylation, etc.), all of which can significantly modify the pI value of a peptide. Peptide mass, as determined by current high mass accuracy mass spectrometers, by itself as the only parameter for peptide identification in large genome databases, is insufficient for high-confidence protein identification and is no longer considered a valid approach to protein identification by the editors of proteomics journals.108 The use of a peptide’s pI value combined with MS/MS data also provides a powerful qualifier for peptide identifications as well as decreasing the peptide search pool. The latter can be achieved by minimizing the number of peptides used to search with by only considering those peptides that are within a discrete pI range in conjunction with the mass range defined by the accuracy of the mass spectrometer used. It is recognized that the 2D liquid-based FFE-IEF/RP-HPLC method described here will play a key role in the analysis of lowMr compounds. In this present study, the ability to fractionate complex mixtures of low-Mr compounds by FFE was demonstrated using a tryptic digest of a cytosolic extract of the human colon cancer cell line LIM 1215. Although the identification of low-Mr compounds in cells and tissues is largely underrepresented in most proteome studies, this class of compounds promises to contain a rich source of previously undiscovered biomarkers of disease.109 ACKNOWLEDGMENT Funding was provided, in part, by the Australian National Health and Medical Research Council under Program Grant 280912. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Note Added after ASAP. The paper was posted on 7/9/04. Labels were added to Figure 6 and the paper was reposted on 7/20/04. Received for review February 19, 2004. Accepted May 21, 2004. AC049717L (105) Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe, C. Proc. Natl. Acad. Sci. U.S.A 1993, 90, 5011-5. (106) Pappin, D. J. C.; Hojrup, P.; Bleasby, A. J. Curr. Biol. 1993, 3, 327-32. (107) Henzel, W. J.; Watanabe, C.; Stults, J. T. J. Am. Soc. Mass Spectrom. 2003, 14, 931-42. (108) Baldwin, M. A. Mol. Cell. Proteomics 2004, 3, 1-9. (109) Liotta, L. A.; Ferrari, M.; Petricoin, E. Nature 2003, 425, 905.

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