Anal. Chem. 2000, 72, 4758-4761
Correspondence
Capillary Isoelectric Focusing without Carrier Ampholytes Tiemin Huang, Xing-Zheng Wu, and Janusz Pawliszyn*
The Guelph-Waterloo Centre for Graduate Work in Chemistry (GWC), Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
A novel capillary isoelectric focusing (CIEF) separation of proteins in pure water was investigated using the wholecolumn imaging detection technique. Protons and hydroxyl ions are produced by the electrolysis of water, and the pH gradient is created by the electromigration of protons and hydroxyl ions into the separation capillary. The addition of acids to the anodic solution and bases to the cathodic solution enhances the formation of the gradients. Fast CIEF separation of proteins by electrolysis of water is demonstrated. CIEF is a high-resolution capillary electrophoresis (CE) technique for the separation of proteins and other zwitterionic compounds with subtle differences in their structures.1,2 Carrier ampholytes (CAs) are commonly used in gel IEF or capillary IEF (CIEF). Because CAs are relatively expensive and troublesome to eliminate in the preparative IEF, can interact with the sample, reduce sensitivity of UV detection, and increase the ion current when coupling CIEF to MS, it would be ideal to conduct CIEF without using CAs. Methods to create a pH gradient by changing the temperature of the buffer,3,4,5 sample autofocusing,6 and steadystate rheoelectrolysis were explored;7 but few satisfactory results were obtained because these methods suffered from a narrow pH range and/or poor separation performance. The focus of this paper is to introduce an ampholyte-free CIEF separation of proteins using the newly developed whole-column imaging detection (WCID) for CIEF. EXPERIMENTAL SECTION Apparatus. An axially illuminated laser-induced fluorescence (LIF) WCID for CIEF, developed in our laboratory and described elsewhere,8 was used for the separation of dilute protein samples. A poly(tetrafluoroethylene) (PTFE) separation capillary (200-µm (1) Righetti, P. G. Isoelectric focusing: theory, methodology and applications; Elsevier Biomedical Press: Amsterdam, 1983; Chapter 1. (2) Kuhn, R.; Hoffsterrer-Kuhn, S. Capillary Electrophoresis: Principles and Practice; Springer Laboratory: Berlin, 1993; Chapter 5. (3) Lochmuller, C. H.; Breiner, S. J. J Chromatogr. 1989, 480, 293-300. (4) Pawliszyn, J.; Wu, J. J. Microcolumn Sep. 1993, 5, 397-401. (5) Fang, X.; Adams, M.; Pawliszyn, J. Analyst 1999, 124, 335-341. (6) Sova, O. J. Chromatogr. 1985, 320, 15-22. (7) Rilbe, H. J. Chromatogr. 1978, 159, 193-205. (8) Huang, T.; Pawliszyn, J. Analyst 2000, 125, 1231-1233.
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i.d. and 6-cm length) was chemically coated inside with hydroxypropylmethylcellulose (HPMC) to eliminate EOF and protein adsorption. The WCID of UV absorbance was conducted in the iCE280 CIEF instrument (Convergent Bioscience Ltd., Toronto, Canada) with a fixed wavelength of 280 nm. A short fused-silica capillary (5.5 cm long) with an i.d. of 100 µm, internally coated with fluorocarbon (J&W Scientific, Folsom, CA), was assembled in a cartridge format (Convergent Bioscience Ltd.) and connected to an eight-port, two-position valve for capillary autoconditioning and sample injection. Methylcellulose (MC) of 0.35% was used to condition the separation capillary for 2 h before conducting the CIEF and was added to the sample to eliminate EOF and protein adsorption. The sample loop was 2.5 µL; however, a 10-µL injection volume was used to ensure there were no air bubbles in the loop. A model A-99 syringe pump (Razel Scientific Instruments, Stamford, CT) was used to deliver the conditioning solution and transfer the sample mixture when the valve was switched from load to inject. The entire process of capillary conditioning, sample injection, data collection, and processing was implemented by a PC computer, and the electropherogram was recorded as absorbance versus the distance to the anode. Materials and Chemicals. Optical fiber with a 100-µm core (FVP100110125) was purchased from Polymicro Technologies Inc. (Phoenix, AZ). Microporous hollow fiber with a pore size of 0.03 µm and 383.3-µm i.d. was obtained from Hoechst Celanese (Frankfurt, Germany). A PTFE capillary of 203-µm i.d. and 406µm o.d. was obtained from Cole-Parmer Instrument Co (Vernon Hills, IL). R-Phycoerythrin was purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA), and green fluorescence protein was obtained as a gift from Convergent Biosciences Ltd. Human hemoglobin control was purchased from Helena Laboratories (Beaumont, TX). MC, HPMC, cytochrome c, and glycerol were obtained from Sigma (St. Louis, MO) and were of analytical grade. Water was purified using an ultrapure water system (Barnstead/ Thermolyne, Dubuque, IA) and was used for all solutions. The sample was prepared by dissolving the proteins in a 20% glycerol solution for the LIF experiment and in a 0.35% methyl cellulose solution for the UV experiment. 10.1021/ac000599l CCC: $19.00
© 2000 American Chemical Society Published on Web 09/01/2000
Figure 1. (a) Color pattern during electrolysis of water. The electrolyte reservoirs and the tubing were all filled with pure water, and methyl red was added as a pH indicator. The anode is to the left, and the cathode is to the right. (b) The schematic for the migration of protons and hydroxyl ions inside the separation capillary.
RESULTS AND DISCUSSION Electrolysis of water is a well-understood electrode reaction, in which protons and hydroxyl ions are produced at the anode and cathode, respectively.9 The electrode reaction in the anode is
2H2O - 4e f 4H+ + O2
(1)
and the electrode reaction in the cathode is
4H2O + 4e f 4OH-+ 2H2
(2)
Electrophoretic migration of ions in the electric field is the basis for the CE technique. We must first consider what will happen if one applies a high voltage to a capillary cartridge whose anode and cathode are connected by a separation capillary that is filled with water. Protons and hydroxyl ions, which are produced by electrolysis, will enter the capillary from the anode end and cathode end of the capillary, respectively. The current along the axis of the capillary is carried by protons and hydroxyl ions since they are the only ions present. Therefore, when high voltage is applied, only the protons and hydroxyl ions can move through the potential gradient. The changes originate at the electrodes and propagate toward the opposite end of the capillary. The migration of protons and hydroxyl ions inside the capillary will increase the proton and hydroxyl ion concentration in the anode and cathode side of the capillary, respectively. Water will be produced when the concentration product of the protons and (9) Zumdahl, S. S. Chemical Principles; D. C. Health and Co.: Toronto, 1992.
Figure 2. CIEF separation of two naturally fluorescent proteins, R-phycoerythrin (3.3 × 10-10 M) and green fluorescent protein (1.8 × 10-8 M), at 500 V/cm. The separation capillary is 200-µm-i.d. PTFE with a length of 6 cm. The samples are directly dissolved in pure water. The anolyte is 100 mM phosphoric acid, and the catholyte is 100 mM sodium hydroxide. An axially illuminated LIF detection system is used.
hydroxyl ions is over the ionization constant of water. The pH is acidic at the anode and basic at the cathode side of the capillary, with a point equal to pH 7 between, yielding a pH gradient along the separation capillary. This phenomenon is easily confirmed experimentally. Figure 1 shows the schematic of electromigration of protons and hydroxyl ions inside the separation capillary and the result of the pH gradient formed by the electrolysis of water. The electrolyte reservoirs and the tubing are all filled with pure water, and methyl red is added as a pH indicator. When power is applied, the color in the anode reservoir and the anode side of the capillary turns red (low pH); while the cathode reservoir and Analytical Chemistry, Vol. 72, No. 19, October 1, 2000
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Figure 3. Dynamic focusing of hemoglobin control (3.1 × 10-6 M) and cytochrome c (6.1 × 10-4 M) at 250 V/cm. The separation capillary cartridge for iCE280 with 100-µm i.d. and a length of 5.5 cm. The separation capillary is conditioned by passing through 0.35% methyl cellulose for 2 h. The samples are mixed with MC (final concentration, 0.35%) in pure water. The anolyte is 100 mM phosphorous acid, and the catholyte is 100 mM sodium hydroxide.
the cathode side of the capillary turns yellow (high pH), with a deeper color for longer electrolysis. The same result is observed when the anodic reservoir is filled with 100 mM phosphoric acid and cathodic reservoir is filled with 100 mM sodium hydroxide. Because water does not have a buffering capacity, and the electrophoretic mobility of a proton is different from that of hydroxyl ion, the pH gradient created by the electrolysis of water may not be linear. However, the possibility arises to utilize this pH gradient for protein separations. This method of pH gradient formation is initially applied to separate dilute protein samples. The fundamental mechanism for the experiment is the same as conventional CIEF. The ampholytes will be protonated in the anodic end and deprotonated in the cathodic end of the capillary and move to cathode or anode, respectively, until their pI points. Figure 2 shows the CIEF separation of R-phycoerythrin (3.3 × 10-10 M) and green fluorescent protein (1.8 × 10-8 M) in the pH gradient created by the electrolysis of water. The focusing is completed within 30 s when 500 V/cm voltage is applied. The pI of R-phycoerythrin is from 4.08 to 4.68,10 and the pI of green fluorescent protein is 5.34.11 From the electropherogram, it is clear that good separation is observed. Table 1 lists the peak positions of both fluorescent proteins under different focusing conditions. Relative standard deviations (RSD) for the peak positions within 5% are obtained, which confirms the good reproducibility of the chemically modified PTFE capillary and the potential applicability of this method. Better RSD values can be expected if the focusing conditions are held constant. Higher protein concentrations are separated by the pH gradient created by the electrolysis of water. For example, human hemoglobin AFSC control (3.1 × 10-6 M) and cytochrome c (6.1 × 10-4 M) are used as target compounds. Figure 3 shows the dynamic focusing of the hemoglobin control and cytochrome c when 250 V/cm voltage is applied in pure water. Proteins with 4760 Analytical Chemistry, Vol. 72, No. 19, October 1, 2000
Table 1. Peak Positions (mm) of the Two Fluorescent Proteins from the Anode under Different Focusing Conditions focusing electric field (V/cm)
focusing time (s)
R-phycoerythrin
green fluorescent protein
40 80 40 40 50 60 30 30 40
33.0 32.0 32.5 33.6 32.8 34.9 31.1 31.6 30.5
36.9 36.2 36.3 37.4 37.5 37.7 35.5 35.5 35.5
average
32.4
36.5
RSD (%)
4.1
2.5
167 167 333 333 333 333 500 500 500
closely related properties, such as the four variants of the human hemoglobin control (A, F, S, C), cannot be separated in this experiment at present. However, proteins with substantially different pIs such as hemoglobin (pI, 7.0) and cytochrome c (pI, 9.5) can be completely separated. In the experiment, faster focusing can be completed within 40 s when higher voltage (500 V/cm) is applied with the focused zone persisting for over 10 min. Proteins focused in pure water have the same pattern as proteins focused in solutions containing carrier ampholytes. It is observed, however, that the distance between peaks is smaller in the pH gradient created by the electrolysis of water, indicating a larger pH span for the gradient created by the electrolysis of water than in the conventional CIEF with pH 3-10 CAs. CIEF in pure water can be accomplished using either water to generate the electrolytes or acid and base as the electrolytes. The presence of protons in the anode reservoir and hydroxyl ions in the cathode (10) Araoz, R.; Lebert, M.; Hader, D. Electrophoresis 1998, 19, 215-219. (11) Righetti, P. G.; Tudor, G. Chromatogr. Rev. 1981, 220, 115-194.
reservoir before focusing accelerates the focusing process; however, this effect needs to be further investigated. The pH gradient in the capillary is not very much affected by the acid and base in the anodic and cathodic reservoirs. pH profiles similar that in Figure 1 are obtained for that case as well. The success of the experiment described above is mainly due to the application of capillaries and whole-column imaging detection. The smaller inner diameter of the capillary format IEF facilitates heat dissipation, which allows for high-voltage application and rapid focusing of proteins. The focusing is monitored in real time, which prevents the disturbance of the pH gradient by the mobilization step in conventional single-point detection CIEF. Without using CAs or buffer compounds, the current in the experiment is so low that higher voltages can be applied, thereby promising even faster CIEF separation. The phenomenon described in this experiment indicates the importance of electromigration of protons and hydroxyl ions to the CIEF separation. In the presence of an electric field, cations and anions will migrate to the anode and cathode, respectively; thus salts in protein samples can be removed from the capillary. Moreover, no MC is needed for chemically modified capillaries; therefore, nearly pure protein zones are obtained in the separation capillary. Without CAs, UV detection below 280 nm becomes
possible (presently, using wavelengths shorter than 260 nm with CAs is impractical), which will lead to a significant improvement in UV detection sensitivity for proteins and peptides. These unique advantages reveal the potential application of this method in coupling with MS, which will greatly simplify the mass spectrum and facilitate protein identification. It is anticipated that the generation of a pH gradient by electrolysis of water in the separation channel will be useful in designing microchip devices, not only for IEF but also for other processes. This approach will also be useful in micropreparative-scale separation of proteins. The anticipated potential problem with this approach would be increased probability of precipitation of proteins compared to the ampholyte techniques. However, since the speed of separation is increased this effect might be minimized. ACKNOWLEDGMENT The authors gratefully acknowledge editorial assistance by Dr. Wayne Mullett and Heather Lord.
Received for review May 23, 2000. Accepted July 19, 2000. AC000599L
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