Reversed‐Phase Isolation of Peptides

William J. Henzel1, John T. Stults1

1 Genentech, Inc., South San Francisco, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.14
DOI:  10.1002/0471142727.mb1014s54
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

In reversed‐phase HPLC, peptides are separated on a hydrophobic stationary phase and eluted with a gradient of increasing organic solvent concentration. Protocols describing the separation of peptides in 5‐ to 500‐pmol quantities via narrow‐bore (2‐mm‐i.d.) or microbore (1‐mm‐i.d.) columns, as well as for the separation of peptides in quantities <5 pmol are provided in this unit. Capillary HPLC columns require a gradient flow rate of 3 to 5 omponents present in a small sample prior to automated sequencing is possible via the procedures for matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry and capillary electrophoresis

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Reversed‐Phase Peptide Separation At the 5‐ to 500‐pmol Level
  • Basic Protocol 2: Reversed‐phase Peptide Separation At ≤5 PMOL
  • Support Protocol 1: Capillary HPLC System Assembly
  • Basic Protocol 3: Peptide Mapping by Matrix‐Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry
  • Alternate Protocol 1: Peptide Mapping by MALDI Mass Spectrometry Using the Matrix Fast‐evaporation Method
  • Basic Protocol 4: Capillary Electrophoresis Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Reversed‐Phase Peptide Separation At the 5‐ to 500‐pmol Level

  Materials
  • Mobile‐phase solvent A: 0.1% (v/v) trifluoroacetic acid (TFA; Pierce) in Milli‐Q water (TFA sold for protein sequencing may not be suitable because some manufacturers add antioxidants that can generate artifacts)
  • Mobile‐phase solvent B: 0.07% to 0.1% (v/v) TFA (Pierce) in acetonitrile or 1‐ or 2‐propanol (Burdick & Jackson or Baker; HPLC‐grade)
  • Solvent modifier: TFA (Pierce)
  • Milli‐Q grade water or equivalent (distilled water is not suitable)
  • Peptide sample
  • HPLC peptide standards, commercial (e.g., PE Biosystems) or tryptic digest (see ), for testing column
  • HPLC system (e.g., Hewlett‐Packard HP‐1090 liquid chromatograph; PE Biosystems model 170A; Michrom BioResources Ultrafast Microprotein Analyzer; Beckman System Gold; Waters Alliance System)
  • recipeDetector flow cell (see recipe)
  • 2‐channel strip‐chart recorder (Kipp & Zonen or equivalent)
  • C18, C8, or C4 reversed‐phase columns, 300 Å, 1‐ or 2‐mm i.d. (e.g., Micra Scientific or Vydac; many other columns from numerous manufacturers can be used)

Basic Protocol 2: Reversed‐phase Peptide Separation At ≤5 PMOL

  Materials
  • Solvent A: 0.1% (v/v) trifluoroacetic acid (TFA) in Milli‐Q water
  • Solvent B: 0.05% to 0.1% (v/v) TFA in acetonitrile
  • Peptide sample
  • Detector flow cell (LC Packings)
  • Strip‐chart recorder (Kipp & Zonen or equivalent)
  • Capillary HPLC system (see protocol 3)
  • Graduated 10‐µl Hamilton glass syringe connected to outlet of flow cell with 0.25‐mm‐i.d. Teflon tubing (LC Packings)
  • Two stopwatches
  • Additional reagents and equipment for reversed‐phase peptide separation at 5 to 500 pmol (see protocol 1) and capillary HPLC system assembly (see protocol 3)

Support Protocol 1: Capillary HPLC System Assembly

  Materials
  • Capillary flow cell (LC Packings)
  • Piston‐ or syringe‐pump splitter (LC Packings) or laboratory‐constructed splitter: 1/16 in. tee (Valco); fingertight fittings; 0.012‐in.‐i.d. sleeve for capillary tubing; fingertight fitting for 0.012‐in. sleeve (Upchurch Scientific); and 150 cm of 0.05‐mm‐i.d., 300‐mm‐o.d. FSC tubing (Polymicro Technologies) or 0.250‐mm‐i.d. Teflon tube inserted into a PEEK fingertight nut and ferrule
  • Capillary pump (PE Biosystems 140‐D) or HPLC piston pump (see protocol 1) for use with an LC Packings splitter, or HPLC syringe pump (PE Biosystems models 120A, 140A, or 170A) for use with a laboratory‐constructed splitter
  • Injector equipped with a 20‐µl loop (Rheodyne)
  • In‐line precolumn filter (Upchurch Scientific)
  • 0.32‐mm × 15‐cm C18 column (LC Packings, Keystone Scientific, Metachem Technologies, Micro‐Tech Scientific, or Michrom Bioresources)

Basic Protocol 3: Peptide Mapping by Matrix‐Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry

  Materials
  • Peptide sample
  • 30% (v/v) acetonitrile/0.1% (v/v) trifluoroacetic acid (TFA)
  • recipeMALDI matrix solution (see recipe)
  • recipeMass standard solution (see recipe)
  • Time‐of‐flight mass spectrometer (e.g., PE Biosystems, Amersham Pharmacia Biotech, Micromass, Brucker, or Kratos)

Alternate Protocol 1: Peptide Mapping by MALDI Mass Spectrometry Using the Matrix Fast‐evaporation Method

  • recipeFast‐evaporation MALDI matrix solution (see recipe)
  • 10% and 0.1% formic acid

Basic Protocol 4: Capillary Electrophoresis Analysis

  Materials
  • 0.1 N NaOH
  • Peptide sample
  • 20 mM sodium phosphate, pH 2.5
  • Capillary electrophoresis (CE) instrument
  • 75‐µm‐i.d. uncoated capillary column with separating length of 50 cm (from manufacturer of CE instrument or Polymicro Technologies)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Aebersold, R. and Morrison, H.D. 1990. Analysis of dilute peptide samples by capillary zone electrophoresis J. Chromatogr. 516:79‐88.
   Beavis, R.C. and Chait, B.T. 1996. Matrix‐assisted laser desorption ionization mass spectronomy of proteins. Methods Enzymol. 270:519‐551.
   Billeci, T.M. and Stults, J.T. 1993. Tryptic mapping of recombinant proteins by matrix‐assisted laser desorption/ionization mass spectrometry. Anal. Chem. 65:1709‐1716.
   Chien, R.L. and Burgi, D.S. 1992. On‐column sample concentration using field amplification in CZE. Anal. Chem. 64:489A‐496A.
   Cobb, K.A. and Novotny, M. 1989. High‐sensitivity peptide mapping by capillary zone electrophoresis and microcolumn liquid chromatography using immobilized trypsin for protein digestion. Anal. Chem. 61:2226‐2231.
   Cobb, K.A. and Novotny, M. 1992. Peptide mapping of complex proteins at the low‐picomole level with capillary electrophoretic separations. Anal. Chem. 64:879‐886.
   Cohen, S.L. and Chait, B.T. 1996. Influence of matrix solution conditions on the MALDI‐MS analysis of peptides and proteins. Anal.Chem. 68:31‐37.
   Davis, M.T. and Lee, T.D. 1992. Analysis of peptide mixtures by capillary high performance liquid chromatography: A practical guide to small‐scale separations. Protein Sci. 1:935‐944.
   Dolan, J.W. 1991. Preventive maintenance and troubleshooting LC instruments. In HPLC of Peptides and Proteins: Separation, Analysis, and Conformation (C.T. Mant and R.S. Hodges, eds.) pp. 23‐30. CRC Press, Boca Raton, Fla.
   Erdjument‐Bromage, H., Geromanos, S., Chodera, A., and Tempst, P. 1993. Successful peptide sequencing with femtomole level PTH‐analysis: A commentary. In Techniques in Protein Chemistry IV (R.H. Angeletti, ed.) pp. 419‐426. Academic Press, San Diego.
   Erdjument‐Bromage, H., Lui, M., Lacomis, L., Grewal, A., Annan, R.S., McNulty, D.E., Carr, S.A., and Tempst, P. 1998. Examination of micro‐tip reversed‐phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis. J. Chromatogr. A. 826:167‐181.
   Figeys, D., Ducret, A., Yates, J.R. III, and Aebersold, R. 1996. Protein identification by solid phase microextraction‐capillary zone electrophoresis‐microelectrospray‐tandem mass spectrometry. BioTechnology 14:1579‐1583.
   Geromanos, S., Casteels, P., Elicone, C., Powell, M., and Tempst, P. 1994. Combined Edman‐chemical and laser‐desorption mass spectrometric approaches to micro peptide sequencing: Optimization and applications. In Techniques in Protein Chemistry V (J.W. Crabb, ed.) pp. 143‐150. Academic Press, San Diego.
   Gevaert, K. and Vandekerckhove, J. 2000. Protein identification methods in proteomics. Electrophoresis 21:1145‐54.
   Gharahdaghi, F., Kirchner, M., Fernandez, J., and Mische, S.M. 1996. Peptide‐mass profiles of polyvinylidene difluoride‐bound proteins by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry in the presence of nonionic detergents. Anal. Biochem. 233:94‐99.
   Gobom, J., Nordhoff, E., Mirgorodskaya, E., Ekman, R., and Roepstorff, P. 1999. Sample purification and preparation technique based on nano‐scale reversed‐phase columns for the sensitive analysis of complex peptide mixtures by matrix‐assisted laser desorption/ionization mass spectrometry. J. Mass Spectrom. 30:105‐16.
   Griffin, P.R., Coffman, J.A., Hood, L.E., and Yates, J.R., III, 1991. Structural analysis of proteins by capillary HPLC electrospray tandem mass spectrometry. Int. J. Mass Spectrum Ion Process. 111:131‐149.
   Grossman, P.D., Wilson, K.J., Petrie, G., and Laura, H.H. 1988. Effect of buffer pH and peptide composition on the selectivity of peptide separations by capillary zone electrophoresis. Anal. Biochem. 173:265‐270.
   Henzel, W.J., Billeci, T.M., Stults, J.T., Wong, S.C., Grimley, C., and Watanabe, C. 1993. Identifying proteins from two‐dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc. Natl. Acad. Sci. U.S.A. 90:5011‐5015.
   Hess, D., Covey, T.C., Winz, R., Brownsey, R.W., and Aebersold, R. 1993. Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis. Protein Sci. 2:1342‐1351.
   Hillenkamp, F., Karas, M., Beavis, R.C., and Chait, R.C. 1991. Matrix‐assisted laser desorption/ionization mass spectrometry of biopolymers. Anal. Chem. 63:1193A‐1203A.
   Huang, E.C. and Henion, J.D. 1991. Packed‐capillary liquid chromatography/ion‐spray tandem mass spectrometry determination of biomolecules. Anal. Chem. 63:732‐739.
   Mant, C.T. and Hodges, R.S. 1990. HPLC of peptides. In HPLC of Biological Macromolecules (K.M. Gooding and F.E. Regnier, eds.) pp. 301‐332. Marcel Dekker, New York.
   Moritz, R.L. and Simpson, R.J. 1992. Application of capillary reversed‐phase high‐performance liquid chromatography to high‐sensitivity protein sequence analysis J. Chromatogr. 599:119‐130.
   Patterson, S.D. and Aebersold, R. 1995. Mass spectrometric approaches for the identification of gel‐separated proteins. Electrophoresis 16:1791‐814.
   Schlabach, T.D. and Wilson, K.J. 1987. Microbore flow‐rates and protein chromatography. J. Chromatogr. 385:65‐74.
   Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. 1996. Mass spectrometric sequencing of proteins from silver‐stained polyacrylamide gels. Anal. Chem. 68:850‐858.
   Tomlinson, A.J., Benson, L.M., Guzman, N.A., and Naylor, S. 1996. Preconcentration and microreaction technology on‐line with capillary electrophoresis. J. Chromatogr. 744:3‐15.
   Vestal, M.L., Juhasz, P., and Martin, S.A. 1995. Delayed extraction matrix‐assisted laser desorption time‐of‐flight mass spectrometry. Rapid Commun. Mass. Spectrom. 9:1044‐1050.
   Wong, S.C., Grimley, C., Padua, A., Bourell, J., and Henzel, W.J. 1993. Peptide mapping of 2‐D gel proteins by capillary HPLC. In Techniques in Protein Chemistry IV (R. H. Angeletti, ed.) pp. 371‐378. Academic Press, San Diego.
Key References
   Davis and Lee, 1992. See above.
  Provides a guide to setting up and using a capillary HPLC.
   Grossman, P.D. and Colburn, J.C. (eds.) 1992. Capillary Electrophoresis—Theory and Practice. Academic Press, San Diego.
  Provides an introduction to capillary electrophoresis.
   Mant, C.T. and Hodges, R.S. (eds.) 1991. HPLC of Peptides and Proteins: Separation, Analysis, and Conformation. CRC Press, Boca Raton, Fla.
  A comprehensive guide to HPLC of peptides and proteins
   Wilm, M. 2000. Mass spectrometric analysis of proteins. Adv. Protein Chem. 54:1‐30.
  A general description of mass spectrometers and their utility for solving problems in protein chemistry.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library