Chemical Cleavage of Proteins in Solution

Dan L. Crimmins1, Sheenah M. Mische2, Nancy D. Denslow3

1 Washington University School of Medicine, St. Louis, Missouri, 2 Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut, 3 University of Florida, Gainesville, Florida
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 11.4
DOI:  10.1002/0471140864.ps1104s40
Online Posting Date:  June, 2005
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Abstract

Described in this unit are five basic protocols that are widely used for specific and efficient chemical cleavage of proteins in solution. Cyanogen bromide (CNBr) cleaves at methionine (Met) residues; BNPS‐skatole cleaves at tryptophan (Trp) residues; formic acid cleaves at aspartic acid‐proline (Asp‐Pro) peptide bonds; hydroxylamine cleaves at asparagine‐glycine (Asn‐Gly) peptide bonds, and 2‐nitro‐5‐thiocyanobenzoic acid (NTCB) cleaves at cysteine (Cys) residues. Because the above loci are at relatively low abundance in most proteins, digestion with these agents will yield relatively long peptides.

     
 
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Table of Contents

  • Basic Protocol 1: Cleavage at the C‐Terminus of Met Residues with CNBr
  • Basic Protocol 2: Cleavage at the C‐Terminus of Trp Residues with BNPS‐Skatole
  • Basic Protocol 3: Cleavage at Asp‐Pro Peptide Bonds with Formic Acid
  • Basic Protocol 4: Cleavage at Asn‐Gly Peptide Bonds with Hydroxylamine
  • Basic Protocol 5: Cleavage at the N‐Terminus of Cys Residues with NTCB
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Cleavage at the C‐Terminus of Met Residues with CNBr

  Materials
  • Lyophilized protein sample in 1.5‐ml capped polypropylene microcentrifuge tube
  • 88% (v/v) formic acid
  • 5 M CNBr in acetonitrile (Aldrich)
  • 10 M NaOH ( appendix 2E)
  • Milli‐Q‐purified water or equivalent
  • Aluminum foil or opaque container
  • 5‐ml glass or polypropylene tube
  • Additional reagents and equipment for reversed‐phase (unit 11.6) or size‐exclusion chromatography (unit 8.3), electrophoresis (unit 10.1), or electroblotting to PVDF membranes (unit 10.7)

Basic Protocol 2: Cleavage at the C‐Terminus of Trp Residues with BNPS‐Skatole

  Materials
  • Lyophilized protein sample
  • Milli‐Q‐purified water or equivalent
  • Dilute acid: e.g., 0.1% trifluoroacetic acid (TFA) or 1% acetic acid or formic acid
  • 1 mg/ml BNPS‐skatole (Pierce; store desiccated at −20°C) in glacial acetic acid (aldehyde‐free; Mallinckrodt Specialty Chemicals), prepared immediately before use
  • Aluminum foil or opaque container
  • Beckman Microfuge 11 equipped with type 13.2 fixed‐angle rotor, or equivalent microcentrifuge
  • Additional reagents and equipment for reversed‐phase (unit 11.6) or size‐exclusion chromatography (unit 8.3), electrophoresis (unit 10.1), or electroblotting to PVDF membranes (unit 10.7)

Basic Protocol 3: Cleavage at Asp‐Pro Peptide Bonds with Formic Acid

  Materials
  • 1 to 50 µg lyophilized protein sample in capped 0.5‐ml microcentrifuge tube
  • 70% (v/v) formic acid prepared immediately before use from 88% formic acid (Fluka)
  • Milli‐Q‐purified water or equivalent
  • Additional reagents and equipment for reversed‐phase (unit 11.6) or size‐exclusion (unit 8.3) chromatography, electrophoresis (unit 10.1), or electroblotting to PVDF membranes (unit 10.7)

Basic Protocol 4: Cleavage at Asn‐Gly Peptide Bonds with Hydroxylamine

  Materials
  • 1 to 50 µg lyophilized protein sample in a 0.5‐ml capped microcentrifuge tube
  • 1.8 M hydroxylamine solution (see recipe)
  • 10% TFA
  • 45°C oven or water bath
  • Additional reagents and equipment for reversed‐phase (unit 11.6) or size‐exclusion chromatography (unit 8.3)

Basic Protocol 5: Cleavage at the N‐Terminus of Cys Residues with NTCB

  Materials
  • Protein samples: 25 to 50 pmol in 20 µl of 200 mM Tris‐acetate, pH 8 (see recipe)/1 mM EDTA/0.1% SDS
  • Ekathiol resin (Ekagen)
  • Nitrogen source
  • Acidified acetone, pH 3, ice‐cold (see recipe)
  • 20 mM NTCB (see recipe)
  • 100 mM sodium borate, pH 9
  • 2 mM NaOH
  • Ultramicrospin columns, gel filtration G10 or G25 media (AmiKa) or acidified acetone, pH 3, ice‐cold (see recipe)
  • 100% acetonitrile
  • 50% and 60% acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA)
  • 0.5% and 0.1% (v/v) TFA
  • 40° and 50°C water baths
  • Centrifuge and rotor (e.g., Eppendorf 5415C, F‐45‐18‐11 rotor)
  • Zip tip (C18‐filled tip; Millipore)
  • Additional reagents and equipment for MALDI‐TOF mass spectrometry (unit 16.2)
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Figures

Videos

Literature Cited

   Aswad, D.W. and Guzzetta, A.W. 1995. Methods for analysis of deamidation and isoasparate formation in peptides and proteins. In Deamidation and Isoaspartate Formation in Peptides and Proteins (D. Aswad, ed.) pp. 7‐29. CRC Press, Boca Raton, Fla.
   Beach, C.M., DeBeer, M.C., Sipe, J.D., Loose, L.D., and DeBeer, F.C. 1992. Human serum amyloid A protein. Complete amino acid sequence of a new variant. Biochem. J. 282:615‐620.
   Catsimpoolas, N. and Wood, J.L. 1966. Specific cleavage of cystine peptides by cyanide. J. Biol. Chem. 241:1790‐1796.
   Crawford, M. 1990. Protein compositions: What is average? ABRF News 1:7.
   Crimmins, D.L., McCourt, D.W., Thoma, R.S., Scott, M.G., Macke, K., and Schwartz, S.D. 1990. In situ chemical cleavage of proteins immobilized to glass fiber and polyvinylidenedifluoride membranes: Cleavage at tryptophan residues with (2‐(2′‐nitrophenylsulfonyl)‐3‐methyl‐3‐bromoindolenine) to obtain internal amino acid sequence. Anal. Biochem. 187:27‐38.
   Daniel, R., Camide, E., Martel, A., LeGoffic, F., Canosa, D., Carrascal, M., and Abian, J. 1997. Mass spectrometric determination of the cleavage sites in Escherichia coli dihydroorotase induced by a cysteine‐specific reagent. Biochemistry 272:26934‐26939.
   Degani, Y. and Patchornick, A. 1974. Cyanylation of sulfhydryl groups by 2‐nitro‐5‐thiocyanobenzoic acid. High yield modification and cleavage of peptides at cysteine residues. Biochemistry 13:1‐11.
   Denslow, N.D. and Nguyen, H.P. 1996. Specific cleavage of blotted proteins at cysteine residues after cyanylation: analysis of products by MALDI‐TOF. In Techniques in Protein Chemistry VII (D.R. Marshak, ed.) pp. 241‐248. Academic Press, San Diego.
   Fontana, A. 1972. Modification of tryptophan with BNPS‐skatole (2‐(2′‐nitrophenylsulfonyl)‐3‐methyl‐3′‐bromoindolenine). Methods Enzymol. 25:419‐423.
   Fontana, A. and Gross, E. 1986. Fragmentation of polypeptides by chemical methods. In Methods in Protein Chemistry: A Handbook (A. Darbre, ed.), pp. 68‐120. John Wiley & Sons, Chichester, U.K., and New York.
   Gardner, A.M., Vaillancourt, R.R., Lange‐Carter, C.A., and Johnson, G.J. 1994. MEK‐1 phosphorylation by MEK kinase, Raf, and mitogen‐activated protein kinase: Analysis of phosphopeptides and regulation of activity. Mol. Biol. Cell 5:193‐201.
   Gross, E. 1967. The cyanogen bromide reaction. Methods Enzymol. 11:238‐255.
   Houghton, R.A. and Li, C.‐H. 1979. Reduction of sulfoxides in peptides and proteins. Anal. Biochem. 98:36‐46.
   Jacobsen, G.R., Schaffer, M.H., Stark, G.R., and Vanaman, T.C. 1973. Specific chemical cleavage in high yield at the amino peptide bonds of cysteine and cystine residues. J. Biol. Chem. 248:6583‐6591.
   Jay, D.G. 1984. A general procedure for the end labeling of proteins and positioning of amino acids in the sequence. J. Biol. Chem. 269:15572‐15578.
   Jue, R.A. and Doolittle, R.F. 1985. Determination of the relative positions of amino acids by partial specific cleavages of end‐labeled proteins. Biochemistry 24:162‐170.
   Kaiser, R. and Metzka, L. 1999. Enhancement of cyanogen bromide cleavage yields for methionyl‐serine and methionyl‐threonine peptide bonds. Anal. Biochem. 266:1‐8.
   Kwong, M.Y. and Harris, R.J. 1994. Identification of succinimide sites in proteins by N‐terminal sequence analysis after alkaline hydroxylamine cleavage. Protein Sci. 3:147‐149.
   Lu, H.S. and Gracy, R.W. 1981. Specific cleavage of glucosephosphate isomerases at cysteinyl residues using 2‐nitro‐5‐thiocyanobenzoic acid: Analyses of peptides eluted from polyacrylamide gels and localization of active site histidyl and lysyl residues. Arch. Biochem. Biophys. 212:347‐359.
   Price, N.C. 1976. Alternative products in the reaction of 2‐nitro‐5‐thiocyanatobenzoic acid with thiol groups. Biochem. J. 159:177‐180.
   Rahali, V. and Gueguen, J. 1999. Chemical cleavage of bovine beta‐lactoglobulin by BNPS‐skatole for preparative purposes: Comparative study of hydrolytic procedures and peptide characterization. J. Prot. Chem. 18:1‐12.
   Smith, B. 1994. Chemical cleavage of proteins. In Methods in Molecular Biology, Vol. 32: Basic Protein and Peptide Protocols (J. Walker, ed.) pp. 297‐309. Humana Press, Totowa, N.J.
   Vestling, M.M., Kelly, M.A., and Fenselau, C. 1995. Optimization by mass spectrometry of a tryptophan‐specific protein cleavage reaction. Rapid Commun. Mass Spectrom. 8:786‐790.
Key References
   Fontana, A. and Gross, E. 1986. See above.
  Excellent source of chemical cleavage procedures.
   Smith, B. 1994. See above.
  Valuable, up‐to‐date summary of several chemical cleavage procedures.
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