Determining the Identity and Structure of Recombinant Proteins

Nancy D. Denslow1, Keith Rose2, Pier Giorgio Righetti3

1 University of Florida, Gainesville, 2 University Medical Center, Geneva, 3 University of Milano, Milan
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 7.3
DOI:  10.1002/0471140864.ps0703s03
Online Posting Date:  May, 2001
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In this unit peptide mapping protocols with separation of the constituent peptides by high‐performance liquid chromatography (HPLC) analysis and by high‐resolution SDS‐PAGE are presented. Peptide mapping is ideally suited for comparative purposes‐‐for example, combined analysis of the recombinant protein and its natural counterpart (or some other well‐characterized standard). This unit also outlines the general strategy used to determine the linkage pattern of a monomeric recombinant protein containing two intramolecular disulfide bonds. The approach is an extension of peptide mapping, where the aim is to isolate and characterize peptides containing only a single disulfide bond. A two‐dimensional electrophoretic method is also described in which the protein isoelectric point is displayed as a function of pH to yield an electrophoretic titration curve. This method is especially useful for checking for deamidation (e.g., of Asn to Asp) in which additional negative charge is introduced into the modified protein.

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Table of Contents

  • Basic Protocol 1: Peptide Mapping by HPLC
  • Alternate Protocol 1: Peptide Mapping by High‐Resolution SDS‐PAGE
  • Support Protocol 1: Digestion of Protein in an SDS‐Polyacrylamide Gel Slice
  • Basic Protocol 2: Determining the Disulfide Linkage Pattern of a Recombinant Protein
  • Basic Protocol 3: Determining Charge Heterogeneity by Two‐Dimensional Titration Curve Analysis
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: Peptide Mapping by HPLC

  • Samples of recombinant and, if possible, native protein
  • Acetone/1 M HCl, −20°C (acidified acetone; optional)
  • 8 M urea/ 50 mM Tris⋅Cl, pH 7.5
  • 45 mM dithiothreitol (DTT)
  • 100 mM iodoacetamide, prepared fresh and kept wrapped in foil
  • Sequencing‐grade protease (unit 11.1) or chemical cleavage agent (unit 11.4): e.g., endoproteinase Lys‐C (Wako Chemicals)
  • recipeDigestion buffer (see recipe)
  • recipeHPLC solvents A and B for peptide mapping (see recipe)
  • 50°C water bath or heating block
  • 15‐ to 25‐cm, 2.5‐mm‐i.d. C18 HPLC column (e.g., Vydac C18, 300 Å, 5‐µm bead)
  • Analytical high‐performance liquid chromatography system
  • Additional reagents and equipment for proteolytic digestion (unit 11.1) and/or chemical cleavage (unit 11.4) of proteins in solution and for digestion in SDS‐polyacrylamide gel (optional; see protocol 3)

Alternate Protocol 1: Peptide Mapping by High‐Resolution SDS‐PAGE

  • 2× SDS sample buffer (unit 10.1)
  • 16 × 16–cm × 1‐mm or 16 × 20–cm × 1‐mm Tris‐tricine slab gel (unit 10.1)
  • 85°C water bath or heating block
  • XAR X‐ray film or equivalent (optional; for detection of radiolabeled proteins, if used)
  • Additional reagents and equipment for radiolabeling of proteins (units 3.3; optional), proteolytic digestion (unit 11.1) and/or chemical cleavage (unit 11.4) of proteins in solution, digestion in SDS‐polyacrylamide gel (optional; see 7.3), one‐dimensional SDS‐PAGE (unit 10.2), and detection of proteins in gels (unit 10.5) or on membranes (unit 10.8)

Support Protocol 1: Digestion of Protein in an SDS‐Polyacrylamide Gel Slice

  • 10 mM Tris⋅Cl (pH 7.5)/ 0.1% (w/v) SDS
  • 1.5‐ml Kontes microcentrifuge tube and pestle, or equivalent
  • 1.5‐ml low‐protein‐binding microfilterfuge tube (0.2‐µm cellulose acetate; Rainin or Costar)
  • Dry ice/ethanol bath (optional)
  • Additional reagents and equipment for one‐ or two‐dimensional SDS‐PAGE (units 10.1 & 10.4), staining with Coomassie blue (unit 10.5), and proteolytic digestion of proteins in gels (unit 11.3)

Basic Protocol 2: Determining the Disulfide Linkage Pattern of a Recombinant Protein

  • Sample of recombinant protein
  • recipeAlkylation buffer (see recipe)
  • Nitrogen or argon, oxygen‐free
  • Guanidine hydrochloride
  • 50 mM and 2 M ammonium bicarbonate
  • recipe2 mg/ml cyanogen bromide in 70% formic acid (see recipe)
  • recipeHPLC solvents A and B for disulfide linkage analysis (see recipe)
  • 1% formic acid
  • Proteases: pepsin, Staphylococcus aureus V8 protease (endoproteinase Glu‐C), and/or trypsin (porcine variety, Sigma)
  • Glacial acetic acid
  • 1 M dithiothreitol (DTT)
  • recipePretreated dialysis tubing with appropriate MWCO (see recipe)
  • Apparatus for reversed‐phase high‐pressure liquid chromatography (RP‐HPLC), preferably with automatic sample injector
  • Appropriate analytical HPLC column: e.g., 25 cm × 4 mm i.d. filled with Nucleosil 5 µm, 300 Å C8 particles (Macherey‐Nagel)
  • Appropriate preparative HPLC column: e.g., 25 cm × 4 cm i.d. filled with Nucleosil 5 µm (as above) for purifying up to ∼0.2 mg protein, or 25 cm × 10 mm i.d. with same packing for up to ∼1.5 mg protein (see for further guidance)
  • Vacuum centrifuge: e.g., Speedvac (Savant)
  • Equipment for mass spectrometry of peptides
  • Additional reagents and equipment for dialysis (unit 4.4; appendix 3B)

Basic Protocol 3: Determining Charge Heterogeneity by Two‐Dimensional Titration Curve Analysis

  • recipeAcrylamide/bisacrylamide stock solution (see recipe)
  • Carrier ampholytes: e.g., Pharmalyte (Pharmacia Biotech), Bio‐Lyte (Bio‐Rad), or Servalyte (Serva)
  • recipe10% (w/v) ammonium persulfate stock solution (see recipe)
  • N,N,N′,N′‐tetramethylethylene diamine (TEMED)
  • Kerosene
  • recipeElectrode solutions (see recipe)
  • Sample of recombinant protein in aqueous solution containing ≤10 mM Tris⋅acetate (or other salt or buffer consisting of weak acids and bases)
  • Coomassie blue staining solution: 0.1% (w/v) Coomassie brilliant blue R‐250/25% (v/v) ethanol/8% (v/v) acetic acid
  • Destaining solution: 25% (v/v) ethanol/8% (v/v) acetic acid
  • Gel Bond PAG sheets
  • Electrophoresis unit for horizontal slab gel operation: e.g., Multiphor (Pharmacia Biotech)
  • Levelling table
  • IEF electrode filter paper strips (Pharmacia Biotech)
  • Constant‐power supply (up to 3000 V, 200 mA, 20 W)
  • Thermostatic water bath: e.g., MultiTemp (Pharmacia Biotech)
  • Titration curve kit (Pharmacia Biotech)
  • Additional reagents and equipment for denaturing IEF in slab gels (unit 10.2)
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Literature Cited

Literature Cited
   Allen, G. 1989. Specific cleavage of the protein in sequencing of proteins and peptides. In Laboratory Techniques in Biochemistry and Molecular Biology,2nd ed. (R.H. Burdon and P.H. van Knippenberg, eds.) pp. 73‐104. Elsevier Science Publishing, New York.
   Arnaud, P., Gianazza, E., Righetti, P.G., and Fudenberg, H.H. 1980. The role of sialic acid in the microheterogeneity of α1 acidic glycoprotein: Study by isoelectric focusing and titration curves. In Electrophoresis '79 (Radola, B.J. ed.) pp. 151‐163. Walter de Gruyter, Berlin.
   Bianchi‐Bosisio, A., Loherlein, D., Snyder, R., and Righetti, P.G. 1980. Protein titration curves by isoelectric focusing‐electrophoresis in highly porous (highly cross‐linked) polyacrylamide gels. J. Chromatogr. 189:317‐330.
   Constans, J., Viau, M., Gouaillard, C., Bouisson, C., and Clerc, A. 1980. Binding affinities between VDBP and vitamin D3,25‐(OH)‐D3, 24‐25‐(OH)2‐D3 and I‐25‐(OH)2‐D3 studied by electrophoretic methods (PAGE‐IEF combined IEF‐electrophoresis) and print‐immunofixation. In Electrophoresis '79 (Radola, B.J., ed.) pp. 701‐710. Walter de Gruyter, Berlin.
   Creighton, T.E. 1979. Electrophoretic analysis of the unfolding of proteins by urea. J. Mol. Biol. 129:235‐264.
   Ek, K. and Righetti, P.G. 1980. Determination of protein ligand dissociation constants and of their pH‐dependence by combined isoelectric focusing electrophoresis (titration curves). Binding of phosphorylases A and B to glycogen. Electrophoresis 1:137‐140.
   Ek, K., Gianazza, E., and Righetti, P.G. 1980. Affino‐titration curves: Determination of dissociation constants of lectin‐sugar complexes and of their pH‐dependence by isoelectric focusing electrophoresis. Biochim. Biophys. Acta 626:356‐365.
   Ellman, G.L. 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82:70‐77.
   Fägerstam, L., Söderberg, L., Wahlström, L., Fredriksson, U.B., Plith, K., and Walldèn, E. 1983. Basic principles used in the selection of monobeads ion exchangers for the separation of biopolymers. Prot. Biol. Fluids 30:621‐628.
   Fernandez, J., DeMott, M., Atherton, D., and Mische, S.M. 1992. Internal protein sequence analysis: Enzymatic digestion for less than 10 µg of protein bound to polyvinylidene difluoride or nitrocellulose membranes. Anal. Biochem. 201:255‐264.
   Gianazza, E., Gelfi, C., and Righetti, P.G. 1980. Isoelectric focusing followed by electrophoresis of proteins for visualizing their titration curves by zymogram and immunofixation. J. Biochem. Biophys. Methods 3:65‐75.
   Glocker, M.O., Arbogast, B., Schreurs, J., and Deinzer, M.L. 1993. Assignment of the inter‐ and intra‐molecular disulfide linkages in recombinant human macrophage colony‐stimulating factor using fast atom bombardment mass spectroscopy. Biochemistry 32:482‐488.
   Harris, R.J., Murnane, A.A., Utter, S.L., Wagner, K.L., Cox, E.T., Polastri, G.D., Helder, J.C., and Sliwkowski, M.B. 1993. Assessing genetic heterogeneity in production cell lines: Detection by peptide mapping of a low level Tyr to Gln sequence variant in a recombinant antibody. Bio/Technology 11:1293‐1297.
   Henner, J. and Sitrin, R.D. 1984. Isoelectric focusing and electrophoretic titration of antibiotics using bioautographic detection. J. Antibiot. 37:1475‐1478.
   Hojrup, P. and Magnusson, S. 1987. Disulphide bridges of bovine Factor X Biochem. J. 245:887‐892.
   Krishnamoorthy, R., Bianchi‐Bosisio, A., Labie, D., and Righetti, P.G. 1978. Titration curves of liganded hemoglobins by combined isoelectric focusing electrophoresis. FEBS Lett. 94:319‐323.
   Jokl, V., Dolejsovà, J., and Matusovà, M. 1979. Zone electrophoresis of organic acids and bases in organic solvents. J. Chromatogr. 172:239‐248.
   Langen, H., Sander, B., Vilbois, F., and Lahm, H.‐W. 1993. Characterization of the proteins c‐kit ligand and DHFR by electrospray mass spectrometry. In Techniques in Protein Chemistry IV (R.H. Angeletti, ed.) pp. 47‐54. Academic Press, San Diego.
   Laskey, R.A. and Mills, A.D. 1975. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56:335‐341.
   Lostanlen, D., Gacon, G., and Kaplan, J.C. 1980. Direct enzyme titration curve of NADH:cytochrome b5 reductase by combined isoelectric focusing/electrophoresis. Interactions between enzyme and cytochrome b5. Eur. J. Biochem. 112:179‐183.
   Markwell, M.A.K. and Fox, C.F. 1978. Surface‐specific iodination of membrane protein of viruses and eucaryotic cells using 1,3,4,6‐tetrachloro‐3α,6α‐diphenylglycoluril. Biochemistry 17:4807‐4817.
   Morris, H.R. and Pucci, P. 1985. A new method for rapid assignment of S‐S bridges in proteins. Biochem. Biophys. Res. Commun. 126:1122‐1128.
   Niekamp, C.W., Hixson, H.F., and Laskowski, M. 1969. Peptide‐bond hydrolysis equilibria in native proteins. Conversion of virgin into modified soybean trypsin inhibitor. Biochemistry 8:16‐22.
   Patterson, S.D. 1994. From electrophoretically separated protein to identification: Strategies for sequence and mass analysis. Anal. Biochem. 221:1‐15.
   Picard, B., Goullet, P., and Krishnamoorthy, R. 1987. A novel approach to study of the structural basis of enzyme polymorphism. Biochem. J 241:877‐881.
   Proudfoot, A.I., Davies, G.D., Turcatti, G., and Wingfield, P.T. 1991. Assignment of the disulfide bridges of unglycosylated recombinant interleukin‐5 expressed in recombinant Escherichia coli using proteolysis at low pH. FEBS Lett. 238:61‐64.
   Rabilloud, T. 1990. The mechanism of protein silver staining in polyacrylamide gels: A 10‐year synthesis. Electrophoresis 11:785‐794.
   Randhawa, Z.I., Witkowska, H.E., Cone, J., Wilkins, J.A., Hughes, P., Yamanishi, K., Yasada, S., Masui, Y., Arthur, P., Kletke, C., Bitsch, F., and Shackleton, C.H.L. 1994. Incorporation of norleucine at methionine positions in recombinant human macrophage stimulating factor (M‐CSF, 4‐153) expressed in Escherichia coli: Structural analysis Biochemistry 3:4352‐4362.
   Riesner, D., Henco, K., and Steger, G. 1991. Temperature gradient gel electrophoresis. In Advances in Electrophoresis (Chrambach, A., Dunn, M.J., and Radola, B.J., eds.) vol. 4 pp. 169‐250. VCH Publishers Weinheim and New York.
   Righetti, P.G., Krishnamoorthy, R., Gianazza, E., and Labie, D. 1978a. Protein titration curves by combined isoelectric focusing‐electrophoresis with hemoglobin mutants as models. J. Chromatogr. 166:455‐460.
   Righetti, P.G., Gacon, G., Gianazza, E., Lostanlen, D., and Kaplan, J.C. 1978b. Titration curves of interacting cytochrome b5 and hemoglobin by isoelectric focusing electrophoresis. Biochem. Biophys. Res. Commun. 85:1575‐1581.
   Righetti, P.G., Menozzi, M., Gianazza, E., and Valentini, L. 1979. Protolytic equilibria of doxorubicin as determined by isoelectric focusing and electrophroetic titration curves. FEBS Lett. 101:51‐55.
   Rose, K., Turcatti, G., Graber, P., Pochon, S., Regamey, P.‐O., Jansen, K.U., Magnenat, E., Aubonney, N., and Bonnefoy, J.‐Y. 1992. Partial characterization of natural and recombinant human soluble CD23. Biochem. J. 286:819‐824.
   Rosengren, A., Bjellqvist, B., and Gasparic, V. 1977. A simple method of choosing optimum pH conditions for electrophoresis. In Electrofocusing and Isotachophoresis (Radola, B.J. and Graesslin, D., eds.) pp. 165‐172. Walter de Gruyter, Berlin.
   Rüchel, R. and Trost, M. 1981. A study of the structural conversions of two carboxyl proteinases employing electrophoresis across a pH gradient. In Electrophoresis '81 (Allen, R.C., and Arnaud, P., eds.) pp. 667‐676. Walter de Gruyter, Berlin.
   Sanger, F. 1959. The chemistry of insulin. Science 129:1340‐1344.
   Schrimsher, J.L., Rose, K., Simona, M.G., and Wingfield, P.T. 1987. Characterization of human and mouse granulocyte‐macrophage‐colony‐stimulating factors derived from Escherichia coli. Biochem. J. 247:195‐199.
   Stahl, E. and Muller, J. 1981. pH‐gradient‐dunnschicht‐chromatographie von benzodiazepinen. J. Chromatogr. 209:484‐488.
   Stults, J.T. 1995. Matrix‐assisted laser desorption/ionization mass spectroscopy (MALDI‐MS). Curr. Opin. Structur. Biol. 5:691‐698.
   Tate, M.E. 1981. Determination of ionization constants by paper electrophoresis. Biochem. J 195:419‐426.
   Thannhauser, T.W., Konishi, Y., and Scheraga, H.A. 1984. Sensitive quantitative analysis of disulfide bonds in polypeptides and proteins. Anal. Biochem. 138:181‐188.
   Thornton, J.M. 1981. Disulfide bonds in globular proteins. J. Mol. Biol. 151:261‐287.
   Troungos, C., Krishnamoorthy, R., Elion, J., and Labie, D. 1982. Titration curves by combined isoelectric focusing‐electrophoresis on a thin layer of agarose gel. 1982. J. Chromatogr. 250:73‐79.
   Valentini, L., Gianazza, E., and Righetti, P.G. 1980. pK determinations via pH‐mobility curves obtained by isoelectric focusing electrophoresis. Theory and experimental verification. J. Biochem. Biophys. Methods 3:323‐338.
   Van Den Oetelaar, P.J. and Hoenders, H.J. 1987. Electrophoretic titration curve in 6 M urea of the bovine eye lens protein α‐crystallin. J. Chromatogr. 411:507‐509.
   Vickers, M.F. and Robinson, P.A. 1983. Protein titration curves using modified cellulose acetate membranes. J. Chromatogr. 275:428‐431.
   Violand, B.N., Schlitter, M.R., Lawson, C.Q., Kane, J.K., Siegal, N.R., Smith, C.E., Kolodziej, E.W., and Duffin, E.L. 1994. Isolation of Escherichia coli synthesized eukaryotic proteins that contain ε‐N‐acetyllysine. Protein Sci. 3:1089‐1097.
   Wingfield, P., Mattaliano, R., MacDonald, R.H., Craig, S., Clore, G.M., Gronenborn, A.M., and Schmeissner, U. 1987. Recombinant‐derived interleukin‐1a stabilized against specific deamidation. Protein Eng. 1:413‐417.
   Wingfield, P., Benedict, R., Turcatti, G., Allet, B., Mermod, J.‐J., DeLamarter, J., Simona, M.G., and Rose, K. 1988. Characterization of recombinant‐derived granulocyte‐colony stimulating factor (G‐CSF). Biochem. J. 256:213‐218.
Key Reference
   Allen, G. 1989. See above.
  Contains excellent information about common proteolytic agents.
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