Anal. Chem. 2003, 75, 215-218
On-Line Hyphenation of Capillary Isoelectric Focusing and Capillary Gel Electrophoresis by a Dialysis Interface Chun Yang, Hechun Liu, Qing Yang, Lingyi Zhang, Weibing Zhang, and Yukui Zhang*
National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116011, China
An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the seconddimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregram is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained. Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) has been a standard technique for high-throughput separation and characterization of biological macromolecules, especially proteins. A main advantage of 2D PAGE is its ability to resolve and investigate the abundance of several thousand proteins in a single sample.1 More than one sample can be dealt with in a single 2D PAGE run, but a total 2D PAGE process usually lasts for at least 1 day and all the manual operations are time-consuming and laborsome. Capillary electrophoresis (CE) is an easier and more rapid alternative to slab electrophoresis. CE was thoroughly reviewed 2-7 and now is a powerful tool for the separation of * Corresponding author: (e-mail)
[email protected]; (phone) +86-4113693427; (fax) +86-411-3693427. (1) Lilley, K. S.; Razzaq, A.; Dupree, P. Curr. Opin. Chem. Biol. 2002, 6, 4650. (2) Gordon, M. J.; Huang, X.; Pentoney, S. L. J.; Zare, R. N. Science 1988, 242, 224-228. (3) Kuhr, W. G.; Monnig, C. A. Anal. Chem. 1992, 64, 389R-407R. (4) Krull, I. S.; Mazzeo, J. R. Nature 1992, 357, 92-94. (5) Monnig, C. A.; Kennedy, R. T. Anal. Chem. 1994, 66, 280R-314R. (6) St. Claire, R. L., III. Anal. Chem. 1996, 68, 569R-586R. (7) Beale, S. C. Anal. Chem. 1998, 70, 279R-300R. 10.1021/ac026187p CCC: $25.00 Published on Web 12/10/2002
© 2003 American Chemical Society
proteins and peptides.8-13 Several kinds of CE mode are built based on different principles. Each single CE mode has its distinct capabilities and limitations. To take full advantage of the capabilities and to avoid the limitations of CE modes it is helpful to combine them one to another 14-22 or with separation methods based on other than electromigration principles. Much attention bas been paid to the hyphenation of CE with liquid chromatography (LC).23-31 There has been a great deal of effort to interface CE with mass spectrometry (MS); nonetheless, MS was considered as a molecular weight detector rather than a separation tool.32-38 (8) Novotny, M. V.; Cobb, K. A.; Liu, J. Electrophoresis 1990, 11, 735-749. (9) Schoneich, C.; Kwok, S. K.; Wilson, G. S.; Rabel, S. R.; Stobaugh, J. F.; Williams, T. D.; Vander Velde, D. G. Anal. Chem. 1993, 65, 67R-84R. (10) Messana, I.; Rossetti, D. V.; Cassiano, L.; Misiti, F.; Giardina, B.; Castagnola, M. J. Chromatogr., B 1997, 699, 149-171. (11) Dolnik, V. Electrophoresis 1997, 18, 2353-2361. (12) Dolnik, V.; Hutterer, K. M. Electrophoresis 2001, 22, 4163-4178. (13) Kasicka, V. Electrophoresis 2001, 22, 4139-4162. (14) Liu, Y.-M.; Sweedler, J. V. Anal. Chem. 1996, 68, 3928-3933. (15) Li, D.; Knobel, H. H.; T. Remcho, V. J. Chromatogr., B 1997, 695, 169174. (16) Hanna, M.; Simpson, C.; Perrett, D. J. Chromatogr., A 2000, 894, 117128. (17) Krivankova, L.; Bocek, P. J. Chromatogr., B 1997, 689 (1), 13-34. (18) Khan, K.; Van Schepdael, A.; Saison-Behmoaras, T.; Van Aerschot, A.; Hoogmartens, J. Electrophoresis 1998, 19, 2163-2168. (19) Dankova, M.; Kaniansky, D.; Fanali, S.; Ivanyi, F. J. Chromatogr., A 1999, 838, 31-43. (20) Kaniansky, D.; Masar, M.; Bielcikova, J.; Ivanyi, F.; Eisenbeiss, F.; Stanislawski, B.; Grass, B.; Neyer, A.; Joehnck, M. Anal. Chem. 2000, 72, 35963604. (21) Gottschlich, N.; Jacobson, S. C.; Culbertson, C. T.; Ramsey, J. M. Anal. Chem. 2001, 73, 2669-2674. (22) Bowerbank, C. R.; Lee, M. L. J. Microcolumn Sep. 2001, 13, 361-370. (23) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 978-984. (24) Lemmo, A. V.; Jorgenson, J. W. J. Chromatogr. 1993, 633, 213-220. (25) Larmann, J. P., Jr.; Lemmo, A. V.; Moore, A. W., Jr.; Jorgenson, J. W. Electrophoresis 1993, 14, 439-447. (26) Lemmo, A. V.; Jorgenson, J. W. Anal. Chem. 1993, 65, 1576-1581. (27) Moore, A. W., Jr.; Jorgenson, J. W. Anal. Chem. 1995, 67, 3448-3455. (28) Lewis, K. C.; Opiteck, G. J.; Jorgenson, J. W.; Sheeley, D. M. J. Am. Soc. Mass Spectrom. 1997, 8, 495-500. (29) Wu, S.-L. Anal. Biochem. 1997, 253, 85-97. (30) Issaq, H. J.; Chan, K. C.; Liu, C.-S.; Li, Q. Electrophoresis 2001, 22, 11331135. (31) Liu, H.; Lin, D.; Yates, J. R., I. BioTechniques 2002, 32, 898, 900, 902, 904, 906, 908, 910-911. (32) Sutton, K.; Sutton, R. M. C.; Caruso, J. A. J. Chromatogr., A 1997, 789, 85-126. (33) Banks, J. F. Electrophoresis 1997, 18, 2255-2266. (34) Wu, J.-T.; Qian, M. G.; Li, M. X.; Zheng, K.; Huang, P.; Lubman, D. M. J. Chromatogr., A 1998, 794, 377-389.
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There is not a single analytical technique in the capillary form such as capillary isoelectric focusing (CIEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary electrochromatography (CEC), or micellar electrokinetic capillary chromatography (MECC) exhibiting comparable resolving power with 2D PAGE based on both isoelectric point (pI) and molecular weight. A multimodal CE method can offer better resolution and peak capacity than a single mode does. CZE is the most common selection incorporated with channel gel electrophoresis,14 electrochromatography,15 and capillary isotachophoresis17-21 in twodimensional (2D) CE systems. In addition, LC itself was coupled to CZE.23-29 In CIEF mode, amphoteric compounds such as proteins are focused in a pH gradient established by carrier ampholytes (CAs) under a high voltage.39 Phosphoric acid is usually used as the anolyte and sodium hydroxide, the catholyte. The anolyte and the catholyte are very different from each other, and they both differ from the buffers used in CEs. As a result, it is hard to combine CIEF with other CE modes. There has been no previous report concerning the hyphenation of CIEF and CGE. In this paper, a dialysis interface was designed and fabricated to integrate CIEF with CGE into an on-line 2D CIEF-CGE system. A hemoglobin (Hb) sample containing diverse variants was separated to check the effectiveness and reliability of the system. The Hb variants were on-line separated in CIEF and CGE modes continuously. It is very rapid to perform 2D CIEF-CGE applying this system. Less than 1 h was needed to complete the separation of an Hb example. EXPERIMENTAL SECTION Materials and Instrumentation. The electrophoresis was performed on a set of TriSep-2000GV from Unimicro Technology (Pleasanton, CA) equipped with a Data Module UV-visible detector (wavelength continuously adjustable) and a CEC Control Module high-voltage source (voltage continuously adjustable). A Workstation Echrom98 of Elite Co. (Dalian, China) was used for data acquisition. The hollow fiber was taken out of a polyethersulfone hollow fiber dialyzer (Diapes-R12, 30-µm i.d., Cawasumi Laboratories, Inc., Tokyo, Japan) for kidney patients. The molecular weight cutoff is 8000. The capillaries (50-µm i.d., 375-µm o.d.) were purchased from Ruiyang Chromatographic Device Co. Ltd. (Yongnian, Hebei, China). N,N,N′,N′-Tetramethylethylenediamine (TEMED, 99%), (3methacryloxypropyl)trimethoxysiliane (γ-MAPS, 98%), and ammonium persulfate (98+%) (all from Acros Organics) and Pharmalyte (pH 3.0-10.0, BioChemika) were used directly without further treatment. Methanol (for HPLC) was purchased from Yucheng Chemical Plant (Shangdong, China). Bovine carbonic anhydrase, rabbit actin, and bovine serum albumin were purchased from Xibasi Biotech Co. (Shanghai, China). Hemoglobin (human) was supplied by Shandong Institute for Drug Control (Jinan, China). It was directly dissolved in the relevant buffers, degassed supersonically for 10 min, and then (35) Zhang, B.; Foret, F.; Karger, B. L. Anal. Chem. 2000, 72, 1015-1022. (36) Choudhary, G.; Apffel, A.; Yin, H.; Hancock, W. J. Chromatogr., A 2000, 887, 85-101. (37) Guzman, N. A.; Stubbs, R. J. Electrophoresis 2001, 22, 3602-3628. (38) Preisler, J.; Hu, P., Rejtar, T.; Moskovets, E.; Karger, B. L. Anal. Chem. 2002, 74, 17-25. (39) Hjerten, S.; Zhu, M. D. J. Chromatogr. 1985, 346, 265-270.
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Figure 1. CIEF of hemoglobin. The capillary is 30 cm long with 22-cm effective length from the anode to detect window. Hb (0.2% w/v) was dissolved in 50 mmol/L Tris-HCl containing Pharmalyte (2% v/v) and TEMED (0.1% v/v). The focusing voltage is 10 kV, and the detection wavelength is 280 nm.
Figure 2. CGE of proteins: (a) carbonic anhydrase (bovine, 31 000), (b) actin (rabbit, 43 000), and (c) albumin (bovine serum, 66 200), all 0.1% (w/v) in CGE buffer. The concentration of Hb is 0.2% (w/v). The length of capillary is 25 cm (18 cm effective). A solution of 50 mmol/L Tris-HCl containing 0.2% (w/v) SDS was used as both the buffer and the electrolytes. Proteins were injected anodically at a voltage of 10 kV for 5 s. The running voltage and the wavelength are the same as in Figure 1.
kept at a temperature of 4 °C. Other reagents are of analytical grade. Fabrication of the Interface. A piece of methacrylate resin plate (50 mm × 30 mm × 4 mm) with a reservoir (5-mm diameter and 3-mm depth) was used as a bolster for the interface (Figure 3). Two sections of Teflon tube (2-mm o.d., 300-µm i.d., and 15mm length) are conglutinated on the opposite sides of the reservoir in the methacrylate plate. Both ends of a hollow fiber (15 mm long) were inserted partly (both ∼5 mm) into the two Teflon tubes, respectively. The slots between the fiber and the tubes were sealed with cyanoacrylate to keep them from leakage. Preparation of Polymer-Coated and Gel-Filled Capillaries. Capillaries are treated routinely, in brief, after being successively
Figure 3. Dialysis interface: (1) methacrylate plate, (2) capillaries, (3) Teflon tubes, (4) hollow fiber, and (5) buffer reservoir.
flushed by 0.5 mol/L HCl, 0.5 mmol/L NaOH, and methanol for 10 min, respectively, and dried in a gas chromatographic (GC) oven at 70 °C for 30 min, the capillary (∼2 m) was filled with a solution of γ-MAPS (50% v/v in methanol) and kept at room temperature with both the ends sealed by silicon septa. About 24 h, unreacted γ-MAPS was washed off using 1 mL of methanol. The capillary was cut into two parts for the purposes of polyacrylamide coating and gel filling, respectively. For polyacrylamide coating, a 10% w/v aqueous solution of acrylamide (containing ammonium persulfate and TEMED, both at a concentration of 1% w/v) was effused in and kept in the capillary for 10 min before its removal. Then the coated capillary was heated in a GC oven at 65 °C for 1 h. An appropriate length of coated capillary must be equilibrated with running buffer before each run of CIEF. In the case of filling the capillary with gel, it was filled with a 10% w/v aqueous solution of acrylamide containing 0.1% w/v sodium dodecyl sulfate (SDS) and 2% w/v TEMED. The capillary was kept at room temperature with both ends immersed in the same solution but with TEMED substituted by ammonium persulfate at the same concentration. A voltage of 20 V/cm was applied for 24 h. The gel-filled capillary should be equilibrated with the CGE buffer at a voltage of ∼100 V/cm for nearly 30 min before use. CIEF and CGE of Proteins. A polyacrylamide-coated capillary was filled with an Hb solution, and the two ends were immersed in 30 mmol/L H3PO4 and 20 mmol/L NaOH, respectively. After a voltage was applied, the focusing began. While the current value declined to ∼15% of its original value, the catholyte (NaOH) was displaced by a solution of 0.1 mol/L NaCl to start cathodic mobilization. The focused protein trains moved toward the cathode and gave signals while they passed the detection window. For CGE experiment, the samples, a solution of Hb and a solution of three molecular weight markers (carbonic anhydrase, actin, albumin), were anodically injected into the gel-filled capillary individually by a voltage for a period of time. After sample injection, the analysis voltage was placed on the capillary to perform CGE. Hyphenation of CIEF and CGE. A polyacrylamide-coated capillary was filled with an Hb sample dissolved in an aqueous solution containing Tris-HCl and CAs. The hollow fiber and Teflon tubes of the interface were also filled with the same solution but without Hb. A gel-filled capillary was previously equilibrated with a Tris-HCl solution with SDS. The two capillaries were inserted into the Teflon tubes as close as possible to the hollow fiber, and they were regarded as the first and second dimensions, respectively (Figure 4). The total procedure should be operated carefully to avoid bubbles in the analysis channel. Simultaneously, the hollow fiber was immerged in a solution of NaOH in the reservoir for the same reason. When a voltage was placed, the focusing started in the first-dimension capillary. After the focusing was finished, the catholyte was replaced with the buffer used in CGE. Another anode was elicited and placed in the anodolyte of the second-dimension capillary, and then a voltage was applied on
Figure 4. Construction of 2D CIEF-CGE framework. The two dimensions of capillary are of the same size and quality used in Figure 1 and Figure 2.
both the dimensions. The CGE buffer was poured to rinse the outer surface of the hollow fiber in the center of the interface during the entire mobilization of the Hb variants. RESULTS AND DISCUSSION Hb is a complex system containing hundreds of known variants.40,41 Apparently a sample not purified sufficiently will magnify the content complexities and separation difficulties. In this way, an Hb sample is a good choice to evaluate the effectiveness and reliability of a comprehensive separation technique. Figure 1 shows the CIEF electrophoregram of an Hb sample. Four peaks stand for the four variants A, F, S, and C, with pIs 7.10, 7.15, 7.25, and 7.50, respectively. The CGE electrophoregram of Hb is shown in Figure 2 . It is compared with three molecular weight markers, bovine carbonic anhydrase, rabbit actin, and bovine serum albumin. At the beginning of the 2D CIEF-CGE performance, Hb variants were focused in the first-dimension capillary and gave four main peaks according to their different pIs. After the replacement of the catholyte by the CGE buffer, the focused protein bands were chemically mobilized toward the interface (cathode). Following the hollow fiber being rinsed by the CGE buffer during the mobilization procedure, there were SDS molecules continuously entering the separation channel in which they were attached to the passing proteins. The negatively charged protein molecules were injected into the gel-filled capillary and further separated in accordance with their sizes or molecular weights. A CIEF band containing proteins with different molecular weights was divided into various bands and gave several peaks in the 2D electrophoregram (Figure 5). Variants A, F, S, and C, but especially A and F, reproducibly give at least three peaks. Hollow fibers are frequently used as microdialyzers for either removal or introduction of small molecules.42-45 An interface (Figure 3) involving a hollow fiber is suitable for incorporating CIEF with CGE to enable a 2D CIEF-CGE implementation. Figure 4 gives a sketch drawing of the 2D CE system. The two dimensions of capillary are connected by a piece of hollow fiber via two sections of Teflon tubes to create an expedite analysis channel. To make the dead volume as small as possible, the inner diameter of the fiber should be equal or close to that of the capillaries. Since silanol groups on the inner surface of the gelfilled capillary were strictly covered, there was no notable (40) Shackleton, C. H. L.; Witkowska, H. E. Anal. Chem. 1996, 68, 29A-33A. (41) Li, M. X.; Liu, L.; Wu, J.-T.; Lubman, D. M. Anal. Chem. 1997, 69, 24512456. (42) Lutz, E. S. M.; Larsson, M. Chromatographia 1999, 49, S28-S34. (43) Wu, J.; Pawliszyn, J. Anal. Chem. 1995, 67, 2010-2014. (44) Lamoree, M. H.; Tjaden, U. R.; van der Greef, J. J. Chromatogr., A 1997, 777, 31-39. (45) Tragas, C.; Pawliszyn, J. Electrophoresis 2000, 21, 227-237.
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Figure 5. 2D CIEF-CGE electrophoregram of Hb. The CIEF capillary is filled with the same Hb solution (without TEMED) in Figure 1 prior to the 2D CE running. The voltage and detection wavelength are the same used for Figure 1. The buffers are the same given in Figure 1 or 2.
electroosmotic flow (EOF) to mobilize the focused analytes in the capillary. In fact, the 2D CIEF-CGE system not only transferred the analytes to the second-dimension capillary but also showed high resolution. This means that 5 mm of bare fiber exposed to the buffer is sufficient for the conveyance of SDS into the analysis channel. A semipermeable hollow fiber lets small molecules traverse freely and obstructs particles with a size up to a certain value. The dialysis interface affords a choice to modify the properties such as pH and ionic strength of the solution in the separation channel. This makes it easy to use a hollow fiber to reduce nonvolatile salt level to adapt to the limited electrolyte tolerance of the ion source in MS.42,46,47 During a 2D LC-CIEF performance, it is necessary to carry out desalting for the eluents from an LC column prior to CIEF performance.43 Simultaneously, the CAs can be introduced into the CIEF capillary with the same hollow fiber interface. The dialysis interface is expected to endow the 2D CIEFCGE with a potential to cooperate with accurate identification tools such as MS. Obviously the cooperation will strengthen the comprehensive resolving power of this 2D CIEF-CGE system. Driven by chemical mobilization, the focused protein bands move slowly in the coated CIEF capillary compared with their negatively charged counterparts in the second dimension (CGE). It allows time long enough for one protein peak to be completely dealt with before another peak enters the CGE capillary. To accomplish optimal separation for each peak in a complex sample, with this 2D CE system it is convenient to adopt simple methods such as monitoring the peaks or reducing or turning off the voltage on the first dimension (CIEF). There are different electric field directions in the two dimensions of capillary. They make molecules with the same kind of charge (either positive or negative) move oppositely. The 2D CIEF-CGE performance would deteriorate if the eluents reentered (46) Hille, J. M.; Freed, A. L.; Watzig, H. Electrophoresis 2001, 22, 4035-4052. (47) Tang, Q.; Harrata, A. K.; Lee, C. S. Anal. Chem. 1997, 69, 3177-3182. (48) Shen, Y.; Berger, S. J.; Anderson, G. A.; Smith, R. D. Anal. Chem. 2000, 72, 2154-2159.
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the CIEF capillary after they are negatively charged in the hollow fiber. Another experiment was carried out to verify the overlapping possibility. As the mobilization began, the UV detector was used to monitor the CIEF capillary at a detection window 7 cm from the interface. The result indicated that no additional peaks different from CIEF were obtained. It is primarily concluded that no negatively charged proteins penetrated into the first-dimension capillary. There are two combinatory forces, cathodic mobilization in the first-dimension capillary and higher voltage gradient in the second-dimension capillary, to prevent the effluents from countermarching back to the first-dimension capillary. The latter were achieved by using a capillary shorter in the second dimension than that in the first dimension. CIEF has the power to concentrate analytes up to ∼540-fold.48 Such a condensed and shortened analyte plug in a capillary is suitable for sample injection to CZE, CGE, MECC, and CEC. It is concluded that CIEF is a proper candidate as the first dimension in a multidimensional CE system. A multidimensional system involving more than two different CE modes could be theoretically realized with this kind of dialysis interface provided that each dimension could be made fast enough to completely separate a peak before the peaks overlap with other peaks coming off the separation. When a high voltage is applied on all capillaries in a multidimensional CE system sharing electrodes, there are alternate electric fields in those capillaries. The electrophoretic forces driving the analytes should be compatible with the alternate directions of the electric fields. For example, as it came to a 3D CIEF-CGE-CZE system originating from the previous 2D system, the anodically mobilized effluents from the CGE capillary could be driven by normal EOF from the anode to the cathode and separated in the third dimension (CZE), which shared an anode with the second dimension (CGE). There would be four electrodes in such a system. CONCLUSIONS The preliminary result indicates that a facile 2D CIEF-CGE system is put to effect using a novel dialysis interface to connect the two dimensions of capillary, maintain the separation voltage, replace the buffer, and preserve the analytes within the separation channel. This 2D CIEF-CGE system is easy to operate with only one high-voltage source and three electrodes. It takes less than 1 h to complete a 2D CIEF-CGE separation for Hb variants. With the dialysis interface, small molecules can be introduced to or removed from the separation channel to change the environment properties of the analytes. Chemical mobilization is an inherent force driving the analytes from the first-dimension capillary to the second one. Potential possibility to achieve multidimensional CE framework and adaptability to other detection tools, i.e., MS, can be extracted from this 2D CE system. ACKNOWLEDGMENT This work was supported by fund (973, 001CB510202) from State Key Fundamental Research (The Ministry of Science and Technology, People’s Republic of China). Finally, the authors thank Shandong Institute for Drug Control for the bounteous protein gifts. Received for review October 1, 2002. Accepted November 12, 2002. AC026187P
