Capillary Electrophoresis of Peptides and Proteins Using Isoelectric Buffers

Pier Giorgio Righetti1, Alessandra Bossi1, Cecilia Gelfi2

1 University of Verona, Verona, 2 Instituto Tecnologie Biomediche Avanzate and Consiglio Nazionale delle Richerce, Milan
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 10.13
DOI:  10.1002/0471140864.ps1013s16
Online Posting Date:  May, 2001
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Abstract

Capillary electrophoresis in acidic isoelectric buffers is a novel methodology allowing fast protein and peptide analysis in uncoated capillaries. Due to the low pH adopted and to the use of dynamic coating with cellulose derivatives, silanol ionization is essentially suppressed and no interaction of macromolecules with the untreated wall ensues. In addition, because of the low conductivity of quasi‚Äźstationary isoelectric buffers, high voltage gradients can be applied (up to 800 V/cm), permitting fast peptide analysis with a high resolving power and minimal diffusional peak spreading.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Analytical Peptide Separations
  • Basic Protocol 2: Separation of Proteins in Acidic Isoelectric Buffers
  • Alternate Protocol 1: Separation of Point Mutations in Polypeptide Chains
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Analytical Peptide Separations

  Materials
  • Peptide mixture (e.g., recipeβ‐casein digestion; see recipe)
  • 0.1 M NaOH
  • Asp buffer: 50 mM aspartic acid (pH = pI = 2.77) containing 0.5% (w/v) hydroxyethyl cellulose (HEC; mol. wt. 27,000 Da, conductivity at 25°C 0.42 mS/cm)
  • Capillary: 37‐cm length (32.4 cm to the detection window); 75‐µm i.d., 375‐µm o.d. (Polymicro Technologies)
  • Capillary zone electrophoresis (CZE) instrument (Bio Focus 3000, Bio‐Rad, or equivalent)

Basic Protocol 2: Separation of Proteins in Acidic Isoelectric Buffers

  Materials
  • Protein sample (e.g., recipegliadin extraction; see recipe)
  • 0.1 M NaOH
  • 70% (v/v) ethanol
  • Asp/urea buffers: 40 mM aspartic acid (pH = pI = 2.77), containing 0.5% (w/v) hydroxyethyl cellulose (HEC; mol. wt. 27,000 Da) and 7 M and 5 M urea (apparent pH 3.9; conductivity at 25°C: 0.7 mS/cm; both values refer to 7 M urea solutions)
  • Capillary: 30‐cm length (25.4 cm to the detection window), 50‐µm i.d., 375‐µm o.d. (Polymicro Technologies)
  • Capillary zone electrophoresis (CZE) instrument (Bio Focus 3000, Bio‐Rad, or equivalent)

Alternate Protocol 1: Separation of Point Mutations in Polypeptide Chains

  • Protein sample (e.g., recipeglobin chain preparation; see recipe)
  • IDA buffer: 50 mM imino diacetic acid (pH = pI = 2.23), containing 0.5% (w/v) hydroxyethyl cellulose (HEC; mol. wt. 27,000 Da) and 7 M and 5 M urea (apparent pH 3.2; conductivity at 25°C: 176 µS/cm)
  • Uncoated capillary, 33‐cm length (26 cm to the detection window), 50‐µm i.d., 375‐µm o.d. (Polymicro Technologies)
  • Capillary zone electrophoresis (CZE) instrument (Bio Focus 3000, Bio‐Rad, or equivalent)
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Figures

Videos

Literature Cited

Literature Cited
   Bossi, A. and Righetti, P.G. 1997. Generation of peptide maps by capillary zone electrophoresis in isoelectric iminodiacetic acid. Electrophoresis 18:2012‐2018.
   Capelli, L., Stoyanov, A.V., Wajcman, H., and Righetti, P.G. 1997. Generation of tryptic maps of α‐ and β‐globin chains by capillary electrophoresis in isoelectric buffers. J. Chromatogr. A 791:313‐322.
   Capelli, L., Forlani, F., Perini, F., Guerrieri, N., Cerletti, P., and Righetti, P.G. 1998. Wheat cultivar discrimination by capillary electrophoresis of gliadins in isoelectric buffers. Electrophoresis 19:311‐318.
   Gelfi, C., Perego, M., and Righetti, P.G. 1996. Capillary electrophoresis of oligonucleotides in sieving liquid polymers in isoelectric buffers. Electrophoresis 17:1470‐1475.
   Hjertèn, S., Valtcheva, L., Elenbring, K., and Liao, J.L. 1995. Fast, high‐resolution (capillary) electrophoresis in buffers designed for high field strengths. Electrophoresis 16:584‐594.
   Mandecki, W. and Hayden, M. 1988. Electrophoresis of DNA fragments in isoelectric histidine buffer. DNA 7:57‐62.
   McLaughlin, G.M., Anderson, K.W., and Hauffe, D.K. 1998. Peptide analysis by capillary electrophoresis: Method development and optimization, sensitivity enhancement strategies and applications. In High‐Performance Capillary Electrophoresis (M.G. Khaledi, ed.) pp. 637‐681. John Wiley & Sons, New York.
   Regnier, F. and Lin, S. 1998. Capillary electrophoresis of proteins. In High‐Performance Capillary Electrophoresis (M.G. Khaledi, ed.) pp. 683‐728. John Wiley & Sons, New York.
   Righetti, P.G. and Nembri, F. 1997. Capillary electrophoresis of peptides in isoelectric buffers. J. Chromatogr.,A 772:203‐211.
   Righetti, P.G., Saccomani, A., Stoyanov, A.V., and Gelfi, C. 1998a. Human globin chain separation by capillary electrophoresis in acidic, isoelectric buffers. Electrophoresis 19:1733‐1737.
   Righetti, P.G., Olivieri, E., and Viotti, A. 1998b. Identification of maize lines via capillary electrophoresis of zeins in isoelectric, acidic buffers. Electrophoresis 19:1738‐1741.
   Terabe, S., Otsuka, K., and Ardo, T. 1985. Micellar electrokinetic chromatography. Anal. Chem. 57:834‐841.
Key References
   Righetti, P.G., Gelfi, C., Perego, M., Stoyanov, A.V., and Bossi, A. 1997. Capillary zone electrophoresis of oligonucleotides and peptides in isoelectric buffers: Theory and methodology. Electrophoresis 18:2145‐2153.
  Three good reviews of both theory and practice for separation of peptides and proteins by CZE in isoelectric buffers.
   Righetti, P.G., Bossi, A., and Gelfi, C. 1997. Capillary isoelectric focusing and isoelectric buffers: An evolving scenario. J. Cap. Elec. 4:47‐59.
  The authors wish to acknowledge the support of grants from the Agenzia Spaziale Italiana (No. ARS‐98‐179) and from MURST (Coordinated Project 40%, Folding/Unfolding of Proteins).
   Stoyanov, A.V. and Righetti, P.G. 1997. Fundamental properties of isoelectric buffers for capillary zone electrophoresis. J. Chromatogr., A 790:169‐176.
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