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Separation of Glycoproteins by Capillary Isoelectric Focusing

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Capillary Electrophoresis of Carbohydrates

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 213))

Abstract

Capillary isoelectric focusing (cIEF) has now become a somewhat routine, accepted mode of operation in capillary electrophoresis (CE). We and others have published several reviews of cIEF (1–14). More and more applications become evident every year, especially from the biotechnology industry (7,15–24). cIEF has become a variation of flat-bed isoelectric focusing (IEF), which is a fairly old, standard method of protein separations. Flat-bed IEF is a form of conventional electrophoresis, which is still often used, especially as part of a two-dimensional (2-D) arrangement involving cIEF-polyacrylamide gel electrophoresis (PAGE) (25–32). Proteomics now employs this 2-D arrangement for complex protein and peptide mixtures, often with immobilized pH gradients (28). In this, we use the terms protein and peptide interchangeably, although they are technically not the same terms. cIEF has some significant advantages over conventional IEF, in that it can be routinely run in a conventional, commercial CE instrument, whether from Bio-Rad, Agilent Technologies, Beckman Coulter, Waters, or others (7,9). It has become a completely automated, analytical method, fully microprocessor controlled, from sample introduction to data readout (9,19–22). A variety of detection methods are possible, such as fixed wavelength ultraviolet (UV), laser-induced fluorescence (LIF), and mass spectrometry (MS) (6,8,16,33–37). It is even possible to utilize a whole column detection approach with UV, and a commercial instrument is now available for such applications (38,39).

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References

  1. Mazzeo, J. and Krull, I. S. (1991) Coated capillaries and additives for the separation of proteins by capillary zone electrophoresis and capillary isoelectric focusing. BioTechniques 10, 638–645.

    PubMed  CAS  Google Scholar 

  2. Mazzeo, J. R., Martineau, J. A., and Krull, I. S. (1992) Performance of isoelectric focusing in uncoated and commercially available coated capillaries. Methods Comp. to Methods Enzymol. 4, 205–212.

    Article  CAS  Google Scholar 

  3. Mazzeo, J. R. and Krull, I. S. (1993) Capillary isoelectric focusing of proteins, peptides, and antibodies, in Capillary Electrophoresis-Technology (Guzman, N., ed.), Marcel Dekker, New York, pp. 795–818.

    Google Scholar 

  4. Mazzeo, J. R., Martineau, J. A., and Krull, I.S. (1993) Peptide mapping using EOF-driven capillary isoelectric focusing. Analyt. Biochem. 208, 323–329.

    Article  PubMed  CAS  Google Scholar 

  5. Mazzeo, J. R., Martineau, J. A., and Krull, I. S. (1994) Advances in the use of coated capillaries for CIEF, in CRC Handbook of Capillary Electrophoresis: Principles, Methods, and Applications (Landers, J. P., ed.), CRC Press, Boca Raton, FL, pp. 495–512.

    Google Scholar 

  6. Liu, X., Sosic, Z., and Krull, I. S. (1996) Capillary isoelectric focusing as a tool in the examination of antibodies, peptides, and proteins of pharmaceutical interest. J. Chromatogr. B 735, 165–190.

    CAS  Google Scholar 

  7. Santora, L. C., Krull, I. S., and Grant, K. (1999) Characterization of recombinant human monoclonal tissue necrosis factor antibody using cation exchange HPLC and capillary isoelectric focusing. Analyt. Biochem. 275, 98–108.

    Article  PubMed  CAS  Google Scholar 

  8. Krull, I. S., Dai, J., Gendreau, C., and Li., G (1997) HPCE methods for the identification and quantitation of antibodies, their conjugates and complexes. J. Pharm. Biomed. Anal. 16, 377–393.

    Article  PubMed  CAS  Google Scholar 

  9. Santora, L. C., Krull, I. S., and Grant, K. L. (1999) Using cIEF to characterize recombinant human monoclonal antibodies. Beckman Coulter P/ACE Setter Newslett. 3, 1–3.

    Google Scholar 

  10. Wehr, T., Rodriguez-Diaz, R., and Zhu, M. (1999) Capillary Electrophoresis of Proteins. Marcel Dekker, New York, pp. 131–233.

    Google Scholar 

  11. Righetti, P. G. Gelfi, C., and Chiari, M. (1996) Isoelectric focusing in capillaries, in Capillary Electrophoresis in Analytical Biotechnology (Righetti, P. G., ed.), CRC Press, Boca Raton, FL, pp. 409–539.

    Google Scholar 

  12. Rodriguez-Diaz, R., Wehr, T., Zhu, M., and Levi, V. (1997) Capillary isoelectric focusing, in Handbook of Capillary Electrophoresis (Landers, J. P., ed.), CRC Press, Boca Raton, FL, pp. 101–138.

    Google Scholar 

  13. Wehr, T., Zhu, M., and Rodriguez-Diaz, R. (1996) Capillary isoelectric focusing, in High Resolution Separation and Analysis of Biological Macromolecules, Part A, Fundamentals (Karger, B. L. and Hancock, W. S., eds.), Academic Press, San Diego, pp. 358–374.

    Chapter  Google Scholar 

  14. Pleasance, T. and Thibault, P. (1993). Interfacing capillary electrophoresis with mass spectrometry, in Capillary Electrophoresis, Theory and Practice (Camilleri, P., ed.), CRC Press, Boca Raton, FL, pp. 311–370.

    Google Scholar 

  15. Hui, J. P. M., Lanthier, P., White, T. C., McHugh, S. G., Yaguchi, M., Roy, R., and Thibault, P. (2001) Characterization of cellobiohydralase I (Ce17A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry. J. Chromatogr. B 1, 349–368.

    Google Scholar 

  16. Shen, Y., Berger, S. J., Anderson, G. A., and Smith, R. D. (2000) High-efficiency capillary isoelectric focusing of peptides. Analyt. Chem. 72, 2154–2159.

    Article  CAS  Google Scholar 

  17. Hagmann, M.-L., Kionka, C., Schreiner, M., and Schwer, C. (1998) Characterization of the F(ab′)2 fragment of a murine monoclonal antibody using capillary isoelectric focusing and electrospray ionization mass spectrometry. J. Chromatogr. A 816, 349–358.

    Article  Google Scholar 

  18. Bowen, S. H. and Schenerman, M. A. (1998) Replacing slab gel electrophoresis methods with capillary electrophoresis for quality control and stability testing. BioPharm November, pp. 42–46.

    Google Scholar 

  19. Moorhouse, K. G., Eusebio, C. A., Hunt, G., and Chen, A. B. (1995) Rapid one-step capillary isoelectric focusing method to monitor charged glycoforms of recombinant human tissue-type plasminogen activator. J. Chromatogr. A 717, 61–69.

    Article  PubMed  CAS  Google Scholar 

  20. Hunt, G., Moorhouse, K. G., and Chen, A. B. (1996) Capillary isoelectric focusing and sodium dodecyl sulfate-capillary gel electrophoresis of recombinant humanized monoclonal antibody HER2. J. Chromatogr. A 744, 295–301.

    Article  PubMed  CAS  Google Scholar 

  21. Schmerr, M. J., Cutlip, R. C., and Jenny, A. (1998) Capillary isoelectric focusing of the scrapie prion protein. J. Chromatogr. A 802, 135–141.

    Article  PubMed  CAS  Google Scholar 

  22. Hunt, G., Totaling, T., and Chen, A. B. (1998) Validation of a capillary isoelectric focusing method for the recombinant monoclonal antibody C2B8. J. Chromatogr. A 800, 355–367.

    Article  PubMed  CAS  Google Scholar 

  23. Tang, S. Anumula, K. R., Maneri, L. R., Nesta, D., and Federici, M. (1998) A method for routine analysis of recombinant immunoglobulins (rIgGs) by capillary isoelectric focusing (cIEF). Paper presented at the Well Characterized Biotechnology Product Pharmaceuticals, San Francisco, CA, January, 1998.

    Google Scholar 

  24. Costello, M. A., Woititz, C., De Feo, J., Stremlo, D., Wen, L.-F. L., Palling, D. J., et al. (1992) Characterization of humanized anti-TAC monoclonal antibody by traditional separation techniques and capillary electrophoresis. J. Liq. Chromatogr. 15, 1081–1097.

    Article  CAS  Google Scholar 

  25. Gordon, A. H. (1969) Electrophoresis of proteins in polyacrylamide and starch gels, in Laboratory Techniques in Biochemistry and Molecular Biology North-Holland, American Elsevier, Amsterdam, Holland.

    Google Scholar 

  26. Laas, T. (1998) Isoelectric focusing, in Protein Purification, Principles, High-Resolution Methods, and Applications (Janson, J.-C. and Ryden, L., eds.), Wiley-Liss, Inc., New York, pp. 495–528.

    Google Scholar 

  27. Bollag, M. D., Rozycki, M. D., and Edelstein, S. J. (1996) Isoelectric focusing and two-dimensional electrophoresis, in Protein Methods 2nd ed., Wiley-Liss, New York, pp. 173–193.

    Google Scholar 

  28. Herbert, B. R., Sanchez, J.-C., and Bini, L. (1997) Two-dimensional electrophoresis: The state of the art and future directions, in Proteome Research: New Fronters in Functional Genomics (Wilkins, M. R., Williams, K. L., Appel, R. D., and Hochstrasser, D. F., eds.), Springer-Verlag, Berlin and Heidelberg, pp. 13–34.

    Google Scholar 

  29. Westermeier, R. (1996) Isoelectric focusing, in Protein Purification Protocols, Methods in Molecular Biology, vol. 59 (Doonan, S., ed.), Humana Press, Totowa, NJ, pp. 239–248.

    Chapter  Google Scholar 

  30. Righetti, P. G., Gelfi, C., and Chiari, M. (1996) Isoelectric focusing in immobilized pH gradients, in High Resolution Separation and Analysis of Biological Macromolecules, Part A, Fundamentals, Methods in Enzymology, vol.270, (Karger, B. L. and Hancock, W. S., eds.), Academic Press, San Diego, CA, pp. 235–254.

    Chapter  Google Scholar 

  31. Protein Electrophoresis, a Technical Manual (1999) Amersham Pharmacia Biotech, Inc., Piscataway, NJ, http://www.apbiotech.com.

  32. 2-D Electrophoresis Using Immobilized pH Gradients, Principles and Methods (1998) Amersham Pharmacia Biotech, Inc., Piscataway, NJ, http://www.apbiotech.com

  33. Strong, R., Liu, H.-J., Cho, B.-Y., Krull, I. S., and Cohen, S.A. (2000) Advantages and limitations of derivatization of peptides for improved performance and detectability in capillary isoelectric focusing (cIEF). J. Liq. Chromatogr. 23, 1775–1807.

    Article  CAS  Google Scholar 

  34. Zhang, C.-X., Xiang, F., Pasa-Tolic, J., Anderson, G. A., Veenstra, T. D., and Smith, R. D. (2000) Step wise mobilization of focused proteins in capillary iso-electric focusing mass spectrometry. Analyt. Chem. 72, 1462–1468.

    Article  CAS  Google Scholar 

  35. Shen, Y., Xiang, F., Veenstra, T. D., Fung, E. N., and Smith, R. D. (1999) High-resolution capillary isoelectric focusing of complex protein mixtures from lysates of microorganisms. Analyt. Chem. 71, 5348–5353.

    Article  CAS  Google Scholar 

  36. Yang, L., Lee, C. S., Hofstadler, S. A., Pasa-Tolic, J. and Smith, R. D. (1998) Capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for protein characterization. Analyt. Chem. 70, 3235–3241.

    Article  CAS  Google Scholar 

  37. Jensen, P. K., Pasa-Tolic, L., Anderson, G. A., Horner, J. A., Lipton, M. S., Bruce, J. E., and Smith, R. D. (1999) Probing proteomes using capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Analyt. Chem. 71, 2076–2084.

    Article  CAS  Google Scholar 

  38. Wang, T. and Hartwick, R. A. (1992) Whole column absorbance detection in capillary isoelectric focusing. Analyt. Chem. 64, 1745–1747.

    Article  CAS  Google Scholar 

  39. Wu, J., Torres, A. R., and Watson, A. H. (1999) Fast IEF analysis of proteins in wide pI range using imaged capillary isoelectric focusing. Paper presented at The Frederick Conference on Capillary Electrophoresis, October, 1999, Frederick, MD.

    Google Scholar 

  40. Krull, I. S., Gendreau, C., and Dai, H. J. (2000) Capillary electrophoresis in combinatorial library analysis, in Analytical Techniques in Combinatorial Chemistry (Swartz, M. E., ed.), Marcel Dekker, New York, pp. 137–172.

    Google Scholar 

  41. Zou, H., Zhang, Y., Lu, P., and Krull, I. S. (1996) Characterization of immunochemical reaction for human growth hormone with its monoclonal antibody by perfusion Protein G affinity chromatography and capillary zone electrophoresis. Biomed. Chromatogr. 10, 78–82.

    Article  PubMed  CAS  Google Scholar 

  42. Weinberger, R. (1993) Practical Capillary Electrophoresis. Academic Press, San Diego, CA.

    Google Scholar 

  43. Weinberger, R. and Lombardi, R. (1997) Method Development, Optimization, and Troubleshooting for High Performance Capillary Electrophoresis. Simon & Schuster. Needham Heights, MA.

    Google Scholar 

  44. eCAP cIEF 3–10 Kit. Isoelectrofocusing of peptides and proteins in the pI range of 3 to 10 by capillary electrophoresis (1997) Beckman Technical Bulletin Beckman Instruments, Inc., Fullerton, CA.

    Google Scholar 

  45. Rodriguez-Diaz, R., Zhu, M., and Wehr, R. (1997) Strategies to improve performance of capillary isoelectric focusing. J. Chromatogr. A 772, 145–160.

    Article  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Chemistry, Northeastern University, Boston, MA, USA

    Ira S. Krull

  2. Quincy, MA, USA

    Sarah Kazmi

  3. Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT, USA

    Huijuan Zhong

  4. Abbott BioResearch Corporation, Worcester Biotech Park, Worcester, MA, USA

    Ling Chen Santora

Authors
  1. Ira S. Krull
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  2. Sarah Kazmi
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  3. Huijuan Zhong
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  4. Ling Chen Santora
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Editor information

Editors and Affiliations

  1. Caprion Pharmaceuticals, Montreal, Quebec, Canada

    Pierre Thibault

  2. Faculty of Pharmaceutical Sciences, Kinki University, Japan

    Susumu Honda

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© 2003 Humana Press Inc.

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Krull, I.S., Kazmi, S., Zhong, H., Santora, L.C. (2003). Separation of Glycoproteins by Capillary Isoelectric Focusing. In: Thibault, P., Honda, S. (eds) Capillary Electrophoresis of Carbohydrates. Methods in Molecular Biology™, vol 213. Humana Press. https://doi.org/10.1385/1-59259-294-5:197

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  • DOI: https://doi.org/10.1385/1-59259-294-5:197

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-826-4

  • Online ISBN: 978-1-59259-294-4

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