Chemical Cleavage of Proteins on Membranes

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 11.5
DOI:  10.1002/0471140864.ps1105s19
Online Posting Date:  May, 2001
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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 bound to membranes. 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. In addition, an describes CNBr cleavage of PVDF‐bound protein previously analyzed by Edman degradation. Finally, a discusses preferred methods of separating and analyzing peptide fragments generated by the chemical cleavage reactions described in the basic protocols.

     
 
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Table of Contents

  • Basic Protocol 1: Cleavage at the C‐terminus of Met Residues with CNBr
  • Alternate Protocol 1: CNBr Cleavage of PVDF‐bound Protein Previously Analyzed by Edman Degradation
  • 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
  • Support Protocol 1: Analysis and Separation of Cleavage Fragments
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

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

  Materials
  • Dried PVDF membrane containing electroblotted protein sample (unit 10.7)
  • recipe0.25 M CNBr in 70% formic acid (see recipe)
  • 10 M NaOH
  • Milli‐Q water or equivalent
  • Clean razor blade
  • Aluminum foil or opaque container
  • 47°C water bath or oven (optional)
  • Additional reagents and equipment for analysis and separation of cleavage fragments (see protocol 7)

Alternate Protocol 1: CNBr Cleavage of PVDF‐bound Protein Previously Analyzed by Edman Degradation

  • recipe1 M CNBr in 70% formic acid (see recipe)
  • 0.5 mg/ml o‐phthalaldehyde (OPA), in butyl chloride (prepared immediately before use)
  • Preconditioned polybrene‐treated glass‐fiber filters

Basic Protocol 2: Cleavage at the C‐terminus of Trp Residues with BNPS‐skatole

  Materials
  • Dried PVDF membrane containing electroblotted protein sample (unit 10.7)
  • Milli‐Q water or equivalent
  • 1 mg/ml BNPS‐skatole (Pierce; store desiccated at −20°C) in 75% glacial acetic acid (aldehyde‐free, Mallinckrodt Specialty Chemicals), prepared immediately before use
  • Clean razor blade
  • Aluminum foil or opaque container
  • 47°C water bath or oven
  • Beckman Microcentrifuge equipped with type 13.2 fixed‐angle rotor, or equivalent microcentrifuge
  • Additional reagents and equipment for analysis and separation of cleavage fragments (see protocol 7)

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

  Materials
  • Dried PVDF membrane containing electroblotted protein sample (unit 10.7)
  • 88% formic acid (Fluka)
  • Preconditioned polybrene‐treated glass‐fiber filter
  • 45°C oven or water bath
  • Additional reagents and equipment for analysis and separation of cleavage fragments (see protocol 7)

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

  Materials
  • Dried PVDF membrane containing electroblotted protein sample (unit 10.7)
  • recipe2 M hydroxylamine solution (see recipe)
  • Milli‐Q water or equivalent
  • 45°C oven or water bath
  • Additional reagents and equipment for analysis and separation of cleavage fragments (see protocol 7)

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

  Materials
  • Dried PVDF membrane containing electroblotted protein sample (unit 10.7).
  • recipeReducing buffer (see recipe)
  • 10 mM 2‐nitro‐5‐thiocyanobenzoic acid (NTCB; Sigma) in 200 mM Tris‐acetate, pH 8 (see recipe for recipeTris‐acetate/EDTA but omit EDTA)
  • Nitrogen source
  • recipe10 mM MES buffer, pH 5 (see recipe)
  • recipe200 mM Tris acetate/1 mM EDTA, pH 8 (see recipe)
  • recipe50 mM sodium borate, pH 9 (see recipe)
  • 60% (v/v) acetonitrile/2.5% (v/v) TFA
  • α‐Cyano‐4‐hydroxycinnamic acid matrix for small fragments or other appropriate matrix (also see unit 16.2)
  • 40° and 50°C water baths
  • Sonicator
  • Additional reagents and equipment for MALDI‐TOF mass spectrometry (unit 16.2)
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Figures

Videos

Literature Cited

   Andrews, P.C. and Dixon, J.E. 1987. A procedure for in situ alkylation of cystine residues on glass fiber prior to protein microsequence analysis. Anal. Biochem. 161:524‐528.
   Aroor, A.R., Denslow, N.D., Singh, L.P., O'Brien, T.W., and Wahba, A.J. 1994. Phosphorylation of rabbit reticulocyte guanine nucleotide exchange factor in vivo. Identification of putative casein kinase II phosphorylation sites. Biochemistry 33:3350‐3357.
   Atherton, D., Fernandez, J., DeMott, M., Andrews, L., and Mische, S.M. 1993a. Routine protein sequence analysis below ten picomoles: One sequencing facility's approach. In Techniques in Protein Chemistry (R.H. Angeletti, ed.) pp. 409‐418. Academic Press, San Diego.
   Atherton, D., Fernandez, J., and Mische, S.M. 1993b. Identification of cysteine residues at the 10 pmol level by carboxamidomethylation of proteins bound to sequencer membrane supports. Anal. Biochem 212:98‐105.
   Bauw, G., Van den Bulcke, M., Van Damme, J., Puype, M., VanMontagu, M., Vandekerckhove, J. 1988. NH2‐terminal and internal microsequencing of proteins electroblotted on inert membranes. In Methods in Protein Sequence Analysis (B. Wittmann‐Liebold, ed.), pp. 220‐233, Springer‐Verlag, Berlin.
   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.
   Brauer, A.W., Oman, C.L., and Margolies, M.N. 1984. Use of o‐phthalaldehyde to reduce background during automated Edman degradation. Anal. Biochem. 137:134‐142.
   Catsimpoolas, N. and Wood, J.L. 1966. Specific cleavage of cystine peptides by cyanide. J. Biol. Chem. 241:1790‐1796.
   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.
   Degani, Y. and Patchornick, A. 1974. Cyanylation of sylfhydryl 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.
   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.
   Jacobson, 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.
   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.
   Landon, M. 1977. Cleavage at aspartyl‐prolyl bonds. Methods Enzymol. 47:145‐149.
   LeGendre, N. 1990. Immobilon P transfer membrane: Applications, utility and protein biochemical analyses. BioTechniques 9:788‐805.
   Liu, Y., Arshavsky, V.Y., and Ruoho, A.E. 1996. Interaction sites of the COOH‐terminal region of the gamma subunit of cGMP phosphodiesterase with the GTP‐bound alpha subunit of transducin. J. Biol. Chem. 271:26900‐26907.
   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.
   Matsudaira, P. 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262:10035‐10038.
   Miczka, G. and Kula, M.R. 1989. The use of polyvinylidene difluoride membranes as blotting matrix in combination with sequence: Application to pyruvate decarboxylase from Zymomonas mobilis. Anal. Lett. 22:2771‐2782.
   Mozdzanowski, J. and Speicher, D.W. 1992. Microsequence analysis of electroblotted proteins. I. Comparison of electroblotting recoveries using different types of PVDF membrane. Anal. Biochem. 207:11‐18.
   Patterson, S.D., Hess, D., Yungwirth, T., and Aebersold, R. 1992. High‐yield recovery of electroblotted proteins and cleavage fragments from a cationic polyvinylidene fluoride‐based membrane. Anal. Biochem. 202:193‐203.
   Price, N.C. 1976. Alternative products in the reaction of 2‐nitro‐5‐thiocyanatobenzoic acid with thiol groups. Biochem. J. 159:177‐180.
   Scott, M.S., Crimmins, D.L., McCourt, D.W., Tarrand, J.J., Eyerman, M.C. and Nahm, M.H. 1988. A simple in situ cyanogen bromide cleavage method to obtain internal amino acid sequence of proteins electroblotted to polyvinylidenedifluoride membranes. Biochem. Biophys. Res. Commun. 155:1353‐1359.
   Scott, M.G., Crimmins, D.L., McCourt, D.W., Zocher, I., Thiebe, R., Zachau, H.G., and Nahm, M.H. 1989. Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. III. A single VkII gene and one of several JK genes are joined by an invariant arginine to form the most common L chain V region. J. Immunol. 143:4110‐4116.
   Scott, M.G., Crimmins, D.L., McCourt, D.W., Chung, G., Schable, K.F., Thiebe, R., Quenzel, E.‐M., Zachau, H.G., and Nahm, M.H. 1991. Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. IV. The less frequently expressed VL are heterogenous. J. Immunol. 147:4007‐4013.
   Simpson, R.J. and Nice, E.C. 1984. In‐situ cyanogen bromide cleavage of N‐terminally blocked proteins in a gas‐phase sequencer. Biochem. Int. 8:787‐791.
   Szewczyk, B. and Summers, D.F. 1988. Preparative elution of proteins blotted to immobilon membranes. Anal. Biochem. 168:48‐53.
   Vaillancourt, R.R., Dhanasekaran, N., and Ruoho, A.E. 1995. The photoactivatable NAD+ analogue [32P]2‐azido‐NAD+ defines intra‐ and inter‐molecular interactions of the C‐terminal domain of G‐protein G alpha t. Biochem. J. 311:987‐993.
   Wadsworth, C.L., Knuth, M.W., Burrus, L.W., Olwin, B.B., and Niece, R.L. 1992. Reusing PVDF electroblotted protein samples after N‐terminal sequencing to obtain unique internal amino acid sequence. In Techniques in Protein Chemistry III (R.H. Angeletti, ed.) pp. 61‐68. Academic Press, San Diego.
Key References
   Fontana, A. and Gross, E. 1986. Fragmentation of polypeptides by chemical methods. In Practical Protein Chemistry: A Handbook (A. Darbre, ed.) pp. 68‐120. John Wiley & Sons, Chichester, U.K., and New York.
  Excellent source of chemical cleavage procedures.
   Matsudaira, P. (ed.) 1989, 1993. A Practical Guide to Protein Purification, Editions 1 and 2. Academic Press, San Diego.
  Various protocols for PVDF‐bound proteins are described.
   Angeletti, R.H. (ed.) 1992, 1993. Techniques in Protein Chemistry, Vols. III and IV. Academic Press, San Diego.
  The above five references are superb compendia of selected, top‐notch papers (presented at the 2nd through 9th Symposia of the Protein Society) related to all areas of protein chemistry, including various solid‐phase PVDF membrane applications.
   Crabb, J. (ed.) 1994, 1995. Techniques in Protein Chemistry, Vols. V and VI. Academic Press, San Diego.
  Valuable, up‐to‐date summary of several chemical cleavage procedures.
   Hugli, T. (ed.) 1998, 1991. Techniques in Protein Chemistry, Vols. I and II. Academic Press, San Diego.
   Marshak, D. 1996, 1997. Techniques in Protein Chemistry, Vol. VII and VIII. Academic Press, San Diego.
   Villafranca, J. (ed.) 1990. Current Protocols in Protein Chemistry: Technique, Structure and Function. Academic Press, San Diego.
   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, Towata, NJ.
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