Comprehensive Protein Profiling by Multiplexed Capillary Zone

Comprehensive Protein Profiling by Multiplexed Capillary Zone Electrophoresis Using Cross-Linked Polyacrylamide Coated Capillaries Shaorong Liu,*,† Lin Gao,† Qiaosheng Pu,† Joann J. Lu,‡ and Xingjia Wang§ Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409, MicroChem Solutions, Lubbock, Texas 79424, and Department of Cell Biology and Biochemistry, Texas Tech University Health Science Center, Lubbock, Texas 79430 Received October 4, 2005

We have recently developed a new process to create cross-linked polyacrylamide (CPA) coatings on capillary walls to suppress protein-wall interactions. Here, we demonstrate CPA-coated capillaries for high-efficiency (>2 × 106 plates per meter) protein separations by capillary zone electrophoresis (CZE). Because CPA virtually eliminates electroosmotic flow, positive and negative proteins cannot be analyzed in a single run. A “one-sample-two-separation” approach is developed to achieve a comprehensive protein analysis. High throughput is achieved through a multiplexed CZE system. Keywords: protein profiling • multiplexed capillary electrophoresis • capillary array electrophoresis • cross-linked polyacrylamide coating

Introduction Capillary electrophoresis (CE) is a powerful analytical tool for protein separations due to its low mass detection limit (and hence the minimal sample consumption) and high resolving power.1-6 Although two-dimensional gel electrophoresis and liquid chromatography are the most frequently used separation techniques, CE is a promising alternative. 1 The commonly used separation modes of CE for protein samples include mainly capillary isoelectric focusing (CIEF),2-8 capillary gel electrophoresis (CGE),9-18 and capillary zone electrophoresis (CZE).19-24 Micellar electrokinetic capillary chromatography (MECC or MEKC)25 and capillary electrochromatography (CEC)26,27 have been used for protein separations, but are not as competitive as CIEF, CGE, or CZE for proteomic analyses.28 CZE is traditionally a high-efficiency separation method,29 and separation efficiencies of 106∼107 plates per meter have been predicted with standard commercial instrumentation.28 On the basis of the separation efficiencies at this level, CZE should have a peak capacity of at least 100.30 In practice, however, few CZE separations of proteins or peptides have demonstrated hundreds of peaks in a single electropherogram. Although CZE has been used to analyze different types of protein samples,31-36 it is mainly limited to separations of less complex mixtures.37-39 A major factor that constrains CZE resolutions is the electrostatic and hydrophobic interactions between protein molecules and capillary walls, and these interactions broaden protein bands. Surface modifications have * To whom correspondence should be addressed. Fax: (806) 742-3210. E-mail: [email protected]. † Department of Chemistry and Biochemistry, Texas Tech University. ‡ MicroChem Solutions. § Department of Cell Biology and Biochemistry, Texas Tech University Health Science Center. 10.1021/pr050335l CCC: $33.50

 2006 American Chemical Society

been proven to be effective to suppress these interactions,40-44 and high-efficiency protein separations are usually obtained using some kind of coated capillaries. There is a semi-inherent problem associated with protein separations by CZE. Because well-coated capillaries are employed for high-resolution protein separations and these capillaries usually have very low electroosmotic flow (EOF), either positively charged or negatively charged (but not all) proteins are electrically driven to the detector for measurements. Since some proteins are positively charged while others are negatively charged under the experimental conditions, only a portion of the proteins are analyzed. Attempts have been made to solve this problem by creating a coating that offers good resolution for protein separations but maintain significant EOF.23,45 Unfortunately, these coatings are unstable in basic (e.g., pH > 7.5) solutions. Recently, we have developed a cross-linked polyacrylamide (CPA) coating for high-resolution protein separations by CIEF8 and CGE.9 In this report, we demonstrate CPA-coated capillaries for the high-efficiency (>2 × 106 plates per meter) separations of proteins by CZE. Because the CPA coating suppresses the EOF almost completely (µeo ≈ 3 × 10-10 m2‚V-1‚s-1 at pH ) 9.2),8 the semi-inherent problem for CZE exists. To overcome this problem, a “one-sample-two-separation” scheme is developed; one separation takes care of the positively charged proteins and the other separation handles the negatively charged proteins. Apparently, this approach will reduce the sample throughput. To compensate and further enhance the throughput, a multiplexed CZE system is built and utilized so that the two separations can be performed simultaneously. In this work, an eight-lane CZE system with UV detection is constructed to demonstrate the feasibility of this approach for high-resolution separation and comprehensive profiling of proteins in complex proteomic samples. Journal of Proteome Research 2006, 5, 323-329

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Figure 1. Schematic diagram of the multiplexed CZE system. (a) Overall configuration. (b) Schematic illustration of the PDA and optical modules assembly. (c) Detailed construction of the optical fiber-photodiode couple.

Experimental Section Reagents and Materials. Cytochrome c (pI: 10.3, MW: 12k), lysozyme (pI: 11.0, MW: 14k), trypsinogen (pI: 9.3, MW: 24k), R-chymotrypsinogen A (pI: 9.1, MW: 25k), myoglobin (pI: 7.1, MW: 17k), R-lactalbumin (pI: 5.1, MW: 14k), trypsin inhibitor (pI: 4.6, MW: 22k), pepsin (pI: 2.8, MW: 35k), and bovine serum albumin (BSA, MW: 66k) were purchased from Sigma (St. Louis, MO). Trypsin was obtained from Promega Corporation (Madison, WI). Acrylamide (AA),N,N′-methylene-bis-acrylamide (Bis), ammonium persulfate (APS) and N,N,N,N′-tetramethyl-ethylenediamine (TEMED) were bought from Bio-Rad Laboratories (Hercules, CA). 2-amino-2-methyl-1-propanol (AMP), cacodylic acid and 3-(Trimethoxysilyl) propyl methacrylate was from Acros Organics (New Jersey, NJ). Fused silica capillaries were purchased from Polymicro Technologies (Phoenix, AZ). Other reagents and chemicals were purchased from Sigma (St. Louis, MO). All solutions were prepared with ultrapure water purified by a NANO pure infinity ultrapure water system (Barnstead, Newton, WA). Instrumentation. All CZE separations were performed on an in-house assembled eight-lane UV absorption CZE system (Figure 1a). Two Glassman (High Bridge, NJ) high voltage power supplies (one positive and one negative) were used in this 324

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experiment. The UV detector part includes three major components, a UV light source, an optical module and a photodiode array (PDA) module. The deuterium lamp light source was directly from an old Waters HPLC system (model 481). Eight fused silica optical fibers were tightly packed together in two rows (four fibers in one row), and affixed on a plastic block which was attached to an x-y-z translation stage. The incident light end of optical fiber bundle was placed near the focus plane of the holographic grating so that all optical fibers were approximately evenly illuminated. No slit was employed before the optical fiber bundle (the cross-section of an optical fiber served as a slit for each fiber). The optimum position of the bundle was determined by maximizing and evenly distributing the light outputs for all eight optical fibers. A detection wavelength of 214 nm was selected to monitor the separated proteins. The optical and PDA modules were made out of black Delrin material. Figure 1b presents an expanded version of the optical and PDA modules. Eight identical parallel grooves (with both width and depth of ∼400 µm) were milled vertically on the inside face (the face in contact with the capillaries) of the optical module to host and secure the capillaries. In the middle of each groove, a pinhole of ∼400 µm in diameter was drilled into the module body for about 2 mm. On the opposite side of the module, a 1.5-mm-diameter hole was drilled perpendicularly toward the 400-µm-hole and stopped at a distance of ∼1 mm to the inside surface. The bottom of this hole was then tapered with a 45° tapered-drill bit. After a 1.0 mm silica ball lens is put in each hole, an optical fiber is inserted in and secured in position with black epoxy glue. In the PDA module, eight photodiodes (Hamamatsu, Japan, model S1226-8BQ) were fixed at the positions in accordance with the eight pinholes in the optical module. Figure 1c presents a cross-section view of the optical and PDA module assembly, looking from the top of a capillary. As the optical and PDA modules were tightened together, the optical fiber, glass ball lens, capillary, and photodiode were lined up. The 214 nm light from the light source was transported by the optical fiber to the silica ball lens, focused onto the capillary, passed through the core of the capillary, and reached the photodiode. Since the photodiode had a photosensitive surface of ∼7 × 7 mm2, most of the transmitted light was received by the photodiode. Meanwhile, when the two modules were held tight together little light can enter the adjacent photodiodes because there was a 15-mm-distance between adjacent photodiodes. The electric signals from all photodiodes were acquired by a NI multifunctional card DAQCard-6062E (National Instruments, Austin, TX) at a sampling rate of 10 Hz and the data were processed with an inhouse developed LabView program. The detailed optimization and characterization of the multiplexed CZE system will be published elsewhere. The multiplex CZE system had eight lanes, one lane was occupied for reference light detection and seven lanes were used for CZE separations. The seven HV reservoirs could be, in any combination, connected to a positive or a negative HV power supply. Referring to Figure 1a, three HV reservoirs were connected to a positive and the other four were linked with a negative HV power supply. The reservoirs at the detection end were grounded. Preparation of CPA Coating. All capillaries for CZE separations were coated with cross-linked polyacrylamide (CPA). Preparation of CPA coating has been described previously.8 Briefly, a bare capillary was first rinsed with 1 M NaOH for 45 min, ultrapure water for 15 min and acetonitrile for 15 min,

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Protein Profiling by Multiplexed Capillary Zone Electrophoresis

Preparation of Crude Protein Extract from MA10 Cells. MA10 cells were cultured following the protocols as described in the literature.46,47 Briefly, the harvested cells were washed three times with ice-cold PBS (phosphate-buffered saline), and lysed in a buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 2 mM PMSF (phenylmethylsulfonyl floride) and 2 µg/mL aprotinine. After 30 s sonication, the solution was centrifuged using Biofuge (Kendro Laboratory Products, Germany) at 13 000 rpm for 8 min. After the supernatant was dialysis desalted in DI water, the solution was vacuum-dried, and re-suspended in water. The final concentration of the crude cell extract has a total protein concentration of ∼5 mg/mL. Preparation of Tryptic Digests of BSA. Trypsin stock solution (10 µg/µL) was prepared by dissolving 200 µg of lyophilized trypsin in 20 µL of 50 mM acetic acid according to the manufacturer’s instructions. This solution was distributed into 10 vials, each containing 2 µL of the stock solution, and stored at -20 °C. 10 mg of BSA was dissolved in 1 mL of 50 mM TrisHCl buffer (pH 8.5) and also stored at -20 °C. For digestion, 50 µL of the BSA solution and 1 µL of trypsin stock solution were mixed and immersed in a 37 °C water bath for about 20 h. This solution was then diluted to 1 mL with ultrapure water before CZE separations.

Figure 2. Electropherograms of the separations of a mixture of four standard proteins in all seven lanes of the multiplexed CZE system. Experimental conditions. Capillaries: 50-µm-i.d., 375µm-o.d., 45-cm-long (∼40-cm-effective length), and CPA-coated, running buffer: 60 mM Tris and 50 mM H3PO4 (pH ) 3.25), separation voltage: +15 kV, sample injection: 5 s at a ∆H ) 10 cm. Peak identifications: 1 - cytochrome c; 2 - lysozyme; 3 trypsinogen; 4 - R-chymotrypsinogen A. Each protein concentration was 0.25 mg/mL. Y-axis full scale was normalized to 10 mAU, and the unit of x-axis was in min.

then dried with helium. A solution containing 0.4% (vol/vol) of 3-(trimethoxysilyl) propyl methacrylate and 0.2% (vol/vol) acetic acid in acetonitrile was flushed through the capillary with a syringe pump at rate of 50 µL/min for 1 h to attach the bifunctional silane reagent to the capillary wall. The capillary was then rinsed with acetonitrile and dried with helium. After 1 mL solution containing 3% (w/v) of acrylamide (AA) and 0.012% (w/v) of N,N′-methylene-bis-acrylamide (Bis) was purged with helium for about 30 min, 1 µL of 10% APS and 1 µL of TEMED were added to the solution through two syringes. This solution was immediately introduced into the bifunctionalized capillary, allowing polymerization reaction to occur inside the capillary. After the reaction proceeded for 8 min, the polymerizing solution was pressurized out and the capillary was flushed with DI water. For immediate use, the capillary was wet-stored (the capillary was filled with water). If the capillary was to be used in a week or longer, the capillary was dry-stored. Drystored capillaries needed to be re-hydrated by soaking the coatings in water overnight before use.

Preparation of the Running Buffers. Four running buffers were used in this experiment. The buffer with a pH of 3.25 was prepared by diluting 1.68 mL of H3PO4 (85%) with ∼300 mL DI water in a 500-mL flask, dissolving 30 mmol of Tris, and filling the flask with water to a final volume of 500 mL. The buffer with a pH of 10.00 was prepared by adding ∼0.2 mL of AMP to 40 mL of 30 mM cacodylic acid. The pH values of the above final solutions were measured to be 3.25 and 10.00. The buffers with pH values of 5.32 and 6.98 were prepared by adjusting the pH of a 50 mM of H3PO4 solutions with 2 M Tris. The final concentration of Tris was ∼70 mM for the pH 5.32 buffer and ∼0.1 M for the pH 6.98 buffer. CZE Separations. The capillaries used in this experiment had a total length of 45 cm (an effective length of ∼40 cm), an inner diameter of 50 µm and an outer diameter of 375 µm. A 3∼5mm-long detection window was made by scraping off the polyimide coating with a razor blade. After a capillary was filled with a proper running buffer, its ends were inserted into the according reservoirs containing with the same solution (∼1.5 mL). For sample introduction, the sampling end of the capillary was taken out of the HV reservoir, dipped into a desired sample vial at a level of 10 cm higher than that of the ground reservoir, maintained there for a given period of time (5∼30 s), and then moved back to the HV reservoir. Immediately after the sample was introduced, a voltage of either +15 kV or -20 kV was applied across the capillary to begin the separation. The absorbance of the separated proteins was monitored at 214 nm. Between runs, the capillary was rinsed with the running buffer solution. The buffer solutions in the anode and cathode reservoirs were replenished after 5∼6 runs.

Table 1. Separation Efficiencies of CZE for Four Standard Proteins no. of theoretical plates per meter, ×106

Cytochrome c lysozeme trypsinogen R-chymotrypsinogen

lane 1

lane 2

lane 3

lane 4

lane 5

lane 6

lane 7

average

1.4 2.3 1.1 1.2

1.8 2.2 1.0 1.3

1.7 2.0 1.2 1.3

1.8 2.1 1.0 1.2

1.7 2.0 1.2 1.3

1.7 1.9 0.9 1.0

1.6 1.9 0.8 1.0

1.7 2.1 1.0 1.2

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Results and Discussion Isolation of the ambient light and prevention of the system vibration are important to reduce the noise level and hence improve the signal-to-noise ratio (S/N) of the UV detector. To isolate the ambient light, the PDA and optical module assembly was wrapped with aluminum foil (or black tape) after seven CPA-coated capillaries were installed (the eighth lane was used as a reference light channel). To prevent the device vibration, the assembly was firmly affixed on two optical posts on an optical table. A detection wavelength of 214 nm was selected because the absorbance at this wavelength is corresponding to the polypeptide bonds of the protein molecules, and an average extinction coefficient of ∼15 L‚g-1‚cm-1 can be used to estimate the protein concentrations.64 High voltages were always applied at the sampling ends of the capillaries, while the detection ends of the capillaries were grounded. Care must be taken when high voltages are used. Note: Even after the HV power supplies are turned off, it takes a few seconds for the residual high voltage to decline to a ground level. The performance of the multiplexed CZE system was evaluated by performing separations of a mixture of standard proteins. The same polarity and field strength were applied for all seven capillaries. Figure 2 presents the separation results from all seven lanes. The signal-to-noise ratio (S/N) varies slightly from lane to lane. In this particular test, the noise level of lane 5 was high due to some random factors. In the later tests, the S/N from lane 5 was comparable to all other lanes. Excellent separation efficiencies were obtained from these CPA-coated capillaries. Table 1 presents the number of theoretical plates per meter from these separation traces presented in Figure 2. For each of the four proteins, the number of theoretical plates exceeded one million. The average plate number for lysozyme reached 2.1 × 106, with the highest plate number of 2.3 × 106 from lane 1. The data presented in Table 1 exceed some of the best results reported previously for CZE separations of proteins.21,22 The results in Figure 2 also exhibit good lane-to-lane migration time reproducibility. The standard deviations of the migration times were 100 000 plates per meter) were also obtained for many of the proteins, and more than 40 well-resolved peaks were observed. The separations were very reproducible (see the fine peak pattern shown in the insets). Because the crude cell extract sample contained all kinds of proteins (large and small, positively charged and negatively charged, hydrophobic and hydrophilic, etc.), these results proved that CPA-coated capillaries are excellent for CZE separations of complex proteomic samples. It is worth mentioning that there should be many more than 40 proteins in the sample. Some of the proteins were undetected due to the limited detection sensitivity of the UV absorbance detector. Some other proteins did not show up because CPA-coated capillary virtually eliminated the EOF8 and most of the negatively charged proteins migrated to the anode reservoir rather than to the detector. Improving the detection sensitivity for CE is an active research area. The current trend seems to be toward the utilization of mass spectrometers (MS) and laser-induced fluorescence (LIF) detectors. Several articles33,49,50 have reviewed the recent progress and research activities in this area. To analyze all proteins, a “one-sample-two-separation” scheme was developed. In one separation, a running buffer with a lower pH and a positive field (from the sampling end to the detection end of the capillary) are used to separate positively charged proteins, and in another separation, a

running buffer with a higher pH and a negative field are used to separate negatively charged proteins. In the first separation at a lower pH, pHL, the proteins with pI > pHL will be positively charged, and these proteins will be separated and detected. In the second separation at a higher pH, pHH, the proteins with pI < pHH will be negatively charged. The negative field will drive these proteins to the detector for measurement. All proteins are thus analyzed by these two separations. In fact, some of the proteins (with pHL< pI < pHH) are analyzed twice, because they are positively charged at pHL and negatively charged at pHH. Apparently, an extended gap between a pHL and a pHH will guarantee a comprehensive protein profile. However, the use of a very low pHL and a very high pHH could adversely affect the coating stability. In addition, the separations may not be optimized at the extreme pH conditions. In optimizing the pHL and pHH, the above factors should be considered. Figure 4 presents an example of adopting the “one-sampletwo-separation” scheme to analyze all proteins in a mixture of eight standard proteins. Using a running buffer with a pHL ) 3.25 and a positive electric field, seven proteins were detected (Figure 4a), but pepsin (pI ) 2.8) was not seen because it was negatively charged under that pH. Employing a running buffer with a pHH ) 10.00 and a negative electric field in the second separation, pepsin and three other proteins showed up in the electropherogram (Figure 4b). From these two separations, all eight proteins were analyzed. Identifications of the peaks were achieved by spiking a protein into the mixture sample and Journal of Proteome Research • Vol. 5, No. 2, 2006 327

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Figure 6. Comprehensive profiling of proteins and peptides in proteomic samples. The crude M10 cell extract contained ∼5 mg/ mL total proteins, and the peptide sample contained ∼0.5 mg/ mL of bovine albumin. +15 kV was applied in lanes 1-3 and -20 kV was used in lanes 4-6. The running buffer with a pH of 5.32 contained 50 mM H3PO4 and ∼70 mM Tris, while that with a pH of 6.98 contained 50 mM H3PO4 and ∼0.1 M Tris. Other buffers and experimental conditions were the same as in Figure 4.

Figure 5. Comprehensive analysis of three synthetic samples composed of standard proteins. Sample 1 contained 1 - lysozyme, 2 - trypsinogen, 3 - R-chymotrypsinogen A, and 4 - pepsin; 0.25 mg/mL each. Sample 2 contained a - cytochrome c; b lysozyme; c - trypsinogen; d - R-lactalbumin; e - R-chymotrypsinogen A and f - pepsin; 0.167 mg/mL each. Sample 3 contained i - myoglobin; ii - R-lactalbumin; iii - trypsin inhibitor and iv - pepsin; 0.25 mg/mL each. For lanes 1-3, the separation conditions were the same as in Figure 4a. For lanes 4-6, the separation conditions were the same as in Figure 4b.

proteins migrated either extremely slowly toward the detector or had migrated in the opposite direction to the cathode reservoir.

comparing the electropherograms from the regular mixture sample and the spiked mixture sample. The selection of a very low pHL and a very high pHH guarantees a comprehensive analysis of all proteins. However, use of such extreme pH solutions is not desired in protecting the CPA coating on the capillary walls. As long as all the proteins can be analyzed, the pHL and pHH are preferred to be close to 7. Figure 4c,d presents the separation results for the sample using a pHL ) 5.32 and a pHH ) 6.98. All eight proteins appeared in the two electropherograms. The lifetime of the coating is expected to be much longer at pH ) 5.32 and 6.98 than at pH ) 3.25 and 10.00. It is important to notice that the migration order of the proteins did not follow their pI values, but agreed to that of the previous report.50 We had expected to see two more peaks in Figure 4b (one for trypsinogen, pI ) 9.3, and the other for R-chymotrypsinogen A, pI ) 9.1), but these peaks did not come out even after 50 min electrophoresis. Likely due to the residual EOF, these

While the “one-sample-two-separation” scheme overcomes the semi-inherent problem of CZE for protein separations, it reduces the sample throughput. To address this issue, a multiplexed CZE system was constructed and used in this work so that the two separations were performed simultaneously. Figure 5 presents the results of separations of three synthetic samples composed of standard proteins, with sample 1 separated in lanes 1 and 4, sample 2 in lanes 2 and 5 and sample 3 in lanes 3 and 6. In lanes 1-3, the running buffer contained 60 mM Tris and 50 mM phosphoric acid (pHL ) 3.25), and a voltage of +15 kV was applied across the separation capillaries. In lanes 4-6, the running buffer contained 50 mM AMP and 30 mM cacodylic acid (pHH ) 10.00), and a voltage of -20 kV was applied across the capillaries. A voltage of -15 kV had been used initially in lanes 4-6, but it was switched to -20 kV later because the output of an old negative HV power supply at -15 kV was not as stable as at -20 kV. Lane 7 was unused. As can be seen from Figure 5, all the proteins in three different samples were analyzed in less than 15 min because the six separations were performed simultaneously. Obviously, the throughput can

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be improved by increasing the number of parallel lanes of the CZE system. To demonstrate the feasibility of this scheme for complex proteomic samples, we prepared two samples: one sample was a crude protein extract from M10 cells,46,47 and the other was a mixture of peptides from trypsin-digested BSA. Figure 6 presents the separation results. Again, +15 kV was applied in lanes 1-3 and -20 kV was used in lanes 4-6. The peptide sample was separated in lanes 3 and 6, and very high resolutions were obtained. In each electropherogram, there were more than 40 sharp peptide peaks. On the basis of these data, a complete peptide mapping can be achieved within 20 min. The proteins in the crude cell extract were separated in lanes 1, 2, 4, and 5, and high resolutions were obtained as well. The positively charged proteins at pHL ) 3.25 and 5.32 were separated in lanes 1 and 2, and the negatively charged proteins at pHH ) 6.98 and pH 10.00 were analyzed in lanes 4 and 5, respectively. Proteins in this sample were comprehensively profiled from a separation in lane 1 or 2 and another separation in lane 4 or 5. The two separations in lanes 2 and 4 may be preferred, because the resolutions are high under these separation conditions and the pH (pHL ) 5.32 and pHH ) 6.98) of the buffer solutions are friendly to the CPA coatings.

Conclusions We have demonstrated CPA-coated capillaries for high efficiency (>2 × 106 plates per meter) CZE separations of proteins in complex proteomic samples. Improvement of the coating chemistry is an ongoing project in our group. A combination of use of improved cross linkers51 and application of surface-confined living radical polymerization52 is expected to further improve the surface coating for capillary electrophoresis. A “one-sample-two-separation” scheme is developed to accomplish high-resolution separations of all proteins, and a multiplexed CZE system is constructed to achieve high throughput protein profiling. There is a lot of potential for this method to be used in proteomic studies, biotech research and drug discoveries, since 96- and 384-lane CZE systems are commercially available. The UV detector may eventually constrain the breadth of the applications of this method, particularly in the area that desires quantifications of proteins at very low concentration levels. Fortunately, this method is compatible with MS detection. Incorporation of this method with MS is anticipated to have a significant impact on proteomic research. In this group, we have an ongoing project to couple MS and CZE with CPA-coated capillaries for protein assays. The results will be published elsewhere.

Acknowledgment. We thank Dr. Mike Harrington at the Huntington Medical Research Institute in Pasadena, CA for valuable discussions and suggestions during the course of this project. References (1) Wittke, S.; Kaiser, T.; Mischak, H. J. Chromatogr. B 2004, 803, 1726. (2) Righetti, P. G.; Bossi, A.; Gelfi, C. J. Capillary Electrophor. 1997, 4, 47-59. (3) Shimura, K. Electrophoresis 2002, 23, 3847-3857. (4) Simpson, D. C.; Smith, R. D. Electrophoresis 2005, 26, 1291-1305. (5) Shen, Y.; Berger, S. J.; Anderson, G. A.; Smith, R. D. Anal. Chem. 2000, 72, 2154-2159. (6) Shen, Y.; Xiang, F.; Veenstra,.T. D.; Fung, E. N.; Smith, R. D. Anal. Chem. 1999, 71, 5348-5353.

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