2-D Capillary Electrophoresis Analysis of Complex Protein Mixture
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1 Application Note: 2-D Capillary Electrophoresis Analysis of Complex Protein Mixture No Rev. B 2004 Target Discovery, Inc. Target Discovery, Inc Fabian Way Palo Alto, CA Tel:
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3 Application Note: No Rev 1.1 Application Challenge: 2-D CE Analyte: Barrett's Esophageal Epithelial Cell Homogenate Product Family: UltraTrol Dynamic Precoatings Product Used: UltraTrol LN 2-D CAPILLARY ELECTROPHORESIS ANALYSIS OF COMPLEX PROTEIN MIXTURE James R. Kraly, Melissa M. Harwood, Emily H. Turner, Norman J. Dovichi Department of Chemistry, University of Washington, Seattle, WA, USA Two-dimensional gel electrophoresis (2-DGE) is routinely the method of choice for the analysis of complex protein mixtures that contain thousands of proteins, such as a cell lysate or tissue homogenate. In most 2-DGE applications, the first dimension separates proteins by their isoelectric points (pi's), while the second dimension resolves proteins by size (MW). 1,2 2-DGE is a high-resolution technique. However, it remains labor intensive, slow, and costly. Furthermore, the resolution of basic proteins, membrane proteins, as well as low and high MW proteins is still technically challenging. 3 In contrast, CE has the advantages of low sample requirements, quick analysis time, online detection, and ease of automation. 4 In recent years, several groups have demonstrated the ability to separate protein or peptide mixtures using multidimensional CE (MDCE ), 5 particularly when the electroosmotic flow is reduced to barely detectable levels. 6 In order to achieve the highest resolution and reproducibility, one needs to control the electroosmotic flow (EOF) throughout the separation process. 6,9 Most frequently, either a statically-coated 7,8 or dynamically-coated capillary is used for EOF regulation 9. Capillaries with static coatings tend not to be coated with 100% efficiency, particularly commercial batches prepared in several meters or tens of meter lots. 9 Moreover, the life time of the coating is often limited, leading to frequent replacements, which are costly. In contrast, dynamic capillary coatings can be applied directly to coat a bare silica capillary each time prior to a separation. Since the coating material is regenerated, better coating efficiency can be achieved when compared to statically coated capillaries. 10 We compare the performance of statically-coated (polyacrylamide, Figure 1) and dynamically-coated (UltraTrol LN, Figure 2) capillaries in the separation of a complex protein mixture obtained from cancerous cell culture using 2-D CE. The first dimension in each example is capillary sieving electrophoresis (CSE). The second dimension is micellar electrokinetic capillary chromatography (MECC) that is conducted in normal polarity with the bare silica capillary (Figure 1) and reverse polarity for the UltraTrol LN coated capillary (Figure 2). The UltraTrol LN dynamic coating appeared to provide better resolution in the capillary sieving electrophoresis (CSE) dimension, allowing more proteins to be resolved in the second dimension. We also demonstrate the reproducibility (Figure 3) of this 2-D CE separation when using UltraTrol LN. In contrast to these results with the dynamic coating, coating erosion forced us to change the static polyacrylamide coated capillary after just a few runs, resulting in elution time irreproducibility from run to run Target Discovery, Inc. All rights reserved 1 Rev. B
4 Figure 1. A 2-D CE separation of esophageal cancer cell homogenate using a polyacrylamide-coated capillary 9 for the first dimension (CSE) and an uncoated capillary for the second dimension (MECC). Separation Buffers: CSE: 5% dextran, 100 mm Ches-Tris, 3.5 mm SDS. MECC: 50 mm Ches-Tris, 15 mm SDS. Separation Conditions: Electrokinetic injection (5 kv, reverse polarity, 5 s). Separation at 20 kv, reverse polarity (CSE); 2 sec fractions, 20 kv, normal polarity (MECC). Capillaries: 30 cm x 30 µm coated (CSE), 27 cm x 30 µm bare silica (MECC). Figure 2. Similar 2-D CE separation of esophageal cancer cell homogenate as shown in Figure 1, however, using capillaries pre-coated with UltraTrol LN for both dimensions and reverse polarity for the MECC. Separation Buffers: CSE: 5% dextran, 100 mm Ches-Tris, 3.5 mm SDS. MECC: 50 mm Ches-Tris, 15 mm SDS. Separation Conditions: Electrokinetic injection (8 kv, reverse polarity, 2 s). Separation at 20 kv, reverse polarity (CSE); 1 sec fractions, 15 kv, reverse polarity (MECC). Capillaries: 20 cm x 30 µm bare silica (CSE), 22 cm x 30 µm bare silica (MECC) Target Discovery, Inc. All rights reserved 2 Rev. B
5 Figure 3. Six replicate runs with UltraTrol LN dynamic pre-coating under the conditions described in Figure 2. 3
6 EXPERIMENTAL Separation Methodology All CE analyses were carried out in a custom-made capillary electrophoresis unit (see descriptions in references 11-12). A voltage program was used to separate peaks in the CSE capillary and then inject them into the MECC capillary. Figure 4 shows how the program operated when UltraTrol LN was used in both capillaries. Capillaries were preconditioned according to the manufacturer s recommendations. In experiments where polyacrylamide coatings were used, the capillaries were rinsed with water for 5 min and separation buffer for 5 min. In experiments were UltraTrol LN was used the capillaries were rinsed with 0.5 M NaOH for 5 min and water for 2 min. The capillaries were then precoated with 1X UltraTrol LN in water, and then rinsed with separation buffer for 2 minutes prior to beginning an analysis. All rinses were done at 10 psi. Sample Preparation Barrett's Esophageal cellular homogenates were first denatured in 1% SDS at 95 C for 4 min. Homogenate proteins were then labeled with 100 nmole of the fluorogenic reagent 3-(2- furoyl)quinoline-2-carboxaldhyde (FQ) in 2.5 mm KCN at 65 C for 5 min. Labeled samples were then diluted to 200 L with the CSE separation buffer (100 mm Ches, 100 mm Tris, 3.5 mm SDS) Figure 4. Voltage program used to generate 2D-CE traces. After injection, a preliminary CSE run for 83 seconds under conditions of reverse polarity was used to generate the first separation. Then the voltage program switched to a repeated sequence of "Fraction Transfer" (1 second), followed by 19-second long "2-D cycle" to generate the second separation. The contents of the CSE capillary were held immobile during the "2D cycle" by eliminating the voltage drop across the capillary. REFERENCES [1] O'Farrel PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem, 1975, 250: [2] Klose J. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutation in mammals. Humangenetik, 1975, 26: [3] Patton WF, Schulenberg B, Steinberg TH. Two-dimensional gel electrophoresis; better than a poke in the ICAT? Curr Opin Biotech, 2002, 13:
7 [4] Weinberger R. Practical capillary electrophoresis, Second Ed. 2000, Academic Press, San Diego, pp [5] Quigley WWC, Dovichi NJ. Capillary electrophoresis for the analysis of biopolymers, Anal Chem, 2004, 76: [6] Schneider, L.V., Hall, M.P., Petesch, R. Protein separation via multidimensional electrophoresis. US (Mar. 25, 2003). [7] Hjerten S. High performance electrophoresis. Elimination of electroendosmosis and solute adsorption. J Chromator, 1985, 347: [8] Cifuentes A, Canalejas P, Diez-Masa JC. Preparation of linear polyacrylamide-coated capillaries. Study of the polymerizatrion process and its effect on capillary electrophoresis performance. J Chromatagr A, 1999, 830: [9] Chang, WWP, Nichols L, Jiang K, Schneider LV. Dynamic coatings for selectable, buffer-insensitive electrophoresis. Am Lab News, 2004, 36:8-13. [10] Horvath J, Dolnik V. Polymer wall coatings for capillary electrophoresis. Electrophoresis, 2001, 22: [11] Michels DA, Hu S, Shoenherr RM, Eggertson MJ, Dovichi NJ. Fully Automated Twodimensional Capillary Electrophoresis for High Sensitivity Protein Analysis. Molec Cell Proteomics, 2002, 1:69-74 [12] Michels DA, Dambrowitz KA, Eggertson MJ, Lauterbach K, Dovichi NJ. Capillary sieving electrophoresis-micellar electrokinetic chromatography fully automated two-dimensional capillary electrophoresis analysis of Deinococcus radiodurans protein homogenate. Electrophoresis, 1004, 25: Target Discovery, Inc. All rights reserved 5 Rev. B
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Application Note: CSE Separation of Cancer Cell Lysate No. 9.200411 Rev. A 2004 Target Discovery, Inc. Target Discovery, Inc. 4015 Fabian Way Palo Alto, CA 94303 Tel: 650-812-8100 www.targetdiscovery.com/eotrol
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