Characterizing Recombinant Proteins Using HPLC Gel Filtration and Mass Spectrometry

Gillian E. Begg1, Sandra L. Harper1, David W. Speicher1

1 The Wistar Institute, Philadelphia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 7.10
DOI:  10.1002/0471140864.ps0710s16
Online Posting Date:  May, 2001
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Abstract

Recombinant proteins are subject to many forms of heterogeneity, including aggregation, proteolytic degradation, chemical modification, mutation, and incorrect translation. This unit describes methods for the detection and identification of these problems using analytical HPLC gel filtration and MALDIā€MS. Preliminary characterization of recombinants is necessary before the structure or function of the protein can be investigated.

     
 
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Table of Contents

  • Basic Protocol 1: HPLC Analysis of Recombinant Proteins
  • Support Protocol 1: Determination of Stokes Radius by HPLC Gel Filtration
  • Support Protocol 2: MALDI‐MS Analysis of Recombinant Proteins
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: HPLC Analysis of Recombinant Proteins

  Materials
  • recipeGel filtration buffer (see recipe)
  • Protein standard (e.g., Bio‐Rad gel filtration standard)
  • Protein sample
  • 0.05% (w/v) sodium azide or 20% (v/v) ethanol
  • Isocratic liquid chromatography system (HPLC or FPLC)
  • Prepacked HPLC or FPLC column (Table 7.10.1)
  • 0.2‐µm filters

Support Protocol 1: Determination of Stokes Radius by HPLC Gel Filtration

  Materials
  • 1 to 10 pmol/µl HPLC‐purified protein sample of interest (see protocol 1)
  • MALDI‐MS‐compatible buffer (e.g., 10 mM ammonium bicarbonate, pH 8)
  • Molecular weight standards
  • recipeSaturated matrix solution (see recipe)
  • Sample target (e.g., gold or stainless steel plate)
  • Matrix‐assisted laser desorption/ionization (MALDI) mass spectrometer (Voyager RP, PerSeptive BioSystems)
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Figures

Videos

Literature Cited

Literature Cited
   Beavis, R.C. and Chait, B.T. 1996. Matrix‐assisted laser desorption ionization mass‐spectrometry of proteins. Methods Enzymol. 270:519‐551.
   Cantor, C.R. and Schimmel, P.R. 1980. Biophysical Chemistry, Part II: Techniques for the Study of Biological Structure and Function. W.H. Freeman, New York.
   Neue, U.D. 1997. HPLC Columns: Theory, Technology, and Practice. John Wiley & Sons, New York.
   Overall, C.M. 1987. A microtechnique for dialysis of small volume solutions with quantitative recoveries. Anal. Biochem. 165:208‐214.
   Schagger, H. 1994. Chromatographic techniques and basic operations in membrane protein purification. In A Practical Guide to Membrane Protein Purification (G.V. Jagow and H. Schagger, eds.) pp. 23‐57. Academic Press, San Diego.
   Seigel, L.M. and Monty, K.J. 1966. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim. Biophys. Acta. 112:346‐362.
Key References
   Beavis and Chait 1996. See above.
  A practical guide to MALDI‐MS of proteins, including details of sample preparation and data analysis.
   Neue, 1997. See above.
  A theoretical and practical guide to HPLC, including properties and selection of columns, methods development, and troubleshooting.
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