Capillary Electrophoresis of Proteins and Peptides

Dean Burgi1, Alan J. Smith2

1 Genomyx, Foster City, California, 2 Stanford University Medical Center, Stanford, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.20
DOI:  10.1002/0471142727.mb1020s54
Online Posting Date:  May, 2001
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Abstract

Capillary electrophoresis (CE) is a high‐resolution technique for the separation of a wide variety of molecules of biological interest such as metabolites, drugs, amino acids, nucleic acids, and carbohydrates. This unit focuses on the use of CE to separate proteins and peptides. As detailed here, CE can also be used to determine the isoelectric point of a protein, either in purified form or in a mixture, by focusing the sample in a pH gradient that is generated within the capillary during electrophoresis. In addition, CE can be used as a micropreparative technique, with either multiple separations that are pooled or a single, larger‐scale separation, for the isolation of peptides from a protease digestion (in much the same way that RP‐HPLC is currently used). In most of these examples the same capillary column can be used for all the separations. Only changes in buffer composition, ionic strength, and the presence or absence of additives are required for each specific application.

     
 
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Table of Contents

  • Instrumentation
  • Separation Theory
  • Strategic Planning
  • Basic Protocol 1: Separation of Proteins by Isoelectric Focusing
  • Basic Protocol 2: Separation of Proteins
  • Basic Protocol 3: Analytical Peptide Separations
  • Basic Protocol 4: Micropreparative Capillary Electrophoresis: Multiple Separations
  • Alternate Protocol 1: Micropreparative Capillary Electrophoresis: Single Separation
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Separation of Proteins by Isoelectric Focusing

  Materials
  • Ampholyte mixture, pH 3 to 10 (Bio‐Rad)
  • Sample containing 0.5 to 1.0 mg protein/ml in water
  • IEF markers (Bio‐Rad; optional)
  • 20 mM sodium hydroxide (NaOH; store at 4°C)
  • 10 mM phosphoric acid (store at 4°C)
  • 50‐µm‐i.d. coated silica capillary column
  • CE instrument (see Table 10.20.1)

Basic Protocol 2: Separation of Proteins

  Materials
  • Sample containing 10 mg protein/ml in water
  • 50 mM and 500 mM sodium borate, pH 8.0 to 9.5 (for separation of proteins with unknown pI)
  • 5 mM and 50 mM buffer at pH >pI (for separation of proteins with known pI; see Table 10.20.2)
  • 50‐µm‐i.d. coated (unknown pI) or uncoated (known pI) fused‐silica capillary column
  • CE instrument (see Table 10.20.1)

Basic Protocol 3: Analytical Peptide Separations

  Materials
  • Peptide mixture: e.g., tryptic digest of β‐lactoglobulin (Applied Biosystems)
  • 0.05 M and 0.25 M sodium phosphate buffer, pH 2.30 (store at 4°C)
  • 0.1 M sodium hydroxide (NaOH)
  • 75‐µm‐i.d. fused‐silica capillary column (Beckman)
  • CE instrument (e.g., P/ACE 5000, Beckman, or equivalent; see Table 10.20.1)

Basic Protocol 4: Micropreparative Capillary Electrophoresis: Multiple Separations

  Materials
  • Peptides: ACTH 4‐10, angiotensin I, and angiotensin II (Sigma)
  • 0.05 mM and 0.25 mM sodium phosphate buffer, pH 2.30 (store at 4°C)
  • 0.1 M sodium hydroxide (NaOH)
  • 75‐µm‐i.d. fused‐silica capillary column (Beckman)
  • CE instrument (e.g., P/ACE 5000, Beckman, or equivalent; see Table 10.20.1)
  • Conical microvials (Beckman)

Alternate Protocol 1: Micropreparative Capillary Electrophoresis: Single Separation

  • 4:1 (v/v) 0.5 M sodium phosphate buffer (pH 2.50)/ethylene glycol
  • 150‐µm‐i.d. fused‐silica capillary column (Polymicro)
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Figures

Videos

Literature Cited

Literature Cited
   Bergman, T. and Jörnvall, H. 1992. Capillary electrophoresis for preparation of peptides and direct determination of amino acids. In Techniques in Protein Chemistry III (R.A. Hogue Angeletti, ed.) pp. 129‐134. Academic Press, San Diego.
   Bullock, J.A. and Yuan, L.‐C. 1991. FSCE of basic proteins in uncoated fused‐silica capillary tubing. J. Microcol. Sep. 3:241‐248.
   Burgi, D.S. and Chien, R.‐L. 1992. On‐column sample concentration using field‐amplification in CZE. Anal. Chem. 64:489A‐495A.
   Chen, F.A., Kelly, L., Palmieri, R., Biehler, R., and Schwartz, H. 1992. Use of high ionic strength buffers for the separation of proteins and peptides with CE. J. Liq. Chromatogr. 15:1143‐1161.
   Dolnik, V., Cobb, K.A., and Novotny, M. 1990. Capillary zone electrophoresis of dilute samples with isotachophoretic preconcentration. J. Microcol. Sep. 2:127‐140.
   Henzel, W.J. and Stults, J.T. 1995. Reversed‐phase isolation of peptides. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, H.L. Ploegh, D.W. Speicher, and P.T. Wingfield, eds.) pp. 11.6.1‐11.6.14. John Wiley & Sons, New York.
   Kenny, J.W., Ohms, J.I., and Smith, A.J. 1993. Micropreparative capillary electrophoresis (MPCE) and micropreparative HPLC of protein digests. In Techniques in Protein Chemistry, IV (R.A. Hogue Angeletti, ed.) pp. 363‐370. Academic Press, San Diego.
   Khaledi, M.G. 1994. Micellar electrokinetic capillary chromatography. In Handbook of Capillary Electrophoresis (J.P. Landers, ed.) pp. 43‐94. CRC Press, Boca Raton, Fla.
   Landers, J.P., Madden, B.J., Oda, R.P., and Spelsberg, T.C. 1992. The use of modifiers of endoosmotic flow for analysis of microheterogeneity. Anal. Biochem. 205:115‐124.
   Mazzeo, J.R. and Krull, I.S. 1992. Improvements in the methods developed for performing isoelectric focusing in uncoated capillaries. J. Chromatogr. 606:291‐296.
   Nielsen, R.G., Richard, E.C., Santa, P.F., Sharknas, D.A., and Sittampalam, G.S. 1991. Separation of antibody‐antigen complexes by CZE, IEF, and HPSEC. J. Chromatogr. 539:177‐185.
   Novotny, M.V., Cobb, K.A., and Liu, J. 1990. Recent advances in CE of proteins, peptides, and amino acids. Electrophoresis 11:735‐749.
   Palmieri, R. and Nolan, J.A. 1994. Protein capillary electrophoresis: Theoretical and experimental considerations for methods development. In Handbook of Capillary Electrophoresis (J.P. Landers, ed.) pp. 325‐368. CRC Press, Boca Raton, Fla.
   Strickland, M. and Strickland, N. 1990. FSCE using phosphate buffer and acidic pH. Am. Lab. 22:60‐65.
   Tran, A.D., Parks, S., Lisi, P.J., Huynh, O.T., Ryall, R.R., and Lane, P.A. 1991. Separations of carbohydrate‐mediated microheterogeneity of human erythropoietin by free solution CE. J. Chromatogr. 542:459‐471.
   Tsuiji, K. and Little, R.J. 1992. Charge‐reversed, polymer‐coated capillary column for the analysis of a recombinant chimeric glycoprotein. J. Chromatogr. 594:317‐324.
   Wiktorowicz, J.E. and Colburn, J.C. 1990. Separation of cationic proteins via charge reversal in CE. Electrophoresis 11:769‐773.
Key References
   McCormick, R.M. 1994. Capillary zone electrophoresis of peptides. In Handbook of Capillary Electrophoresis (J.P. Landers, ed.) pp. 287‐324. CRC Press, Boca Raton, Fla.
  Excellent review of both theory and practice for separation of peptides by CE.
   Palmieri and Nolan, 1994. See above.
  Good review of methods development for protein separations.
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