Cyanogen bromide (CNBr) peptide mapping
Cyanogen bromide and formic acid peptide mapping Protocol from:
Simplified sample recovery from CNBr
Skopp, R.N. and Lane, L.C. Fingerprinting of proteins cleaved in solution by cyanogen bromide. Appl. and Theoret. Electrophoresis 1, 61-64 (1989)
Restriction endonucleases cleave DNA into large, discrete fragments which show characteristic gel electrophoretic patterns. The order of restriction fragments in the DNA serves as a framework for nucleotide sequence analysis. Specific chemical cleavage could be similarly useful for characterizing plant virus capsid proteins.
Chemical cleavage methods can produce large fragments with defined ends. Gross and Witkop [J. Biol. Chem. 237, 1856 (1962), Methods in Enzymology 11, 238 (1967)] introduced cyanogen bromide (CNBr) cleavage of met-X and Piszkiewicz,et al [Biochem. Biophys. Res. Commun. 40, 1173 (1970)] introduced formic acid cleavage of asp-pro. Unlike restriction endonucleases these reagents yield complex mixtures of partial and complete digestion products. Separation patterns are not so easy to interpret as for restriction fragments.
1.7% of peptide bonds are met-X and 0.25% are asp-pro (Creighton, Proteins, Structures and Molecular Properties. W.H.Freeman, 1984, p.7). These bonds are "randomly" distributed; some proteins have too few to be useful and others have many and give complex, difficult-to-interpret patterns. Protein chemists commonly produce CNBr fragments for amino acid sequence analysis. Cleavage is often sufficient so that protein chemists consider it "complete".
Standard CNBr digestion involves reduction, alkylation, dialysis and lyophilization. Proteins are then dissolved in 70% formic acid and incubated with CNBr. After another lyophilization, fragments are dissolved in buffer and analyzed by gel filtration, or more recently by gel electrophoresis.
We have developed rapid methods that avoid lyophilization and dialysis, permitting simultaneous CNBr or formic acid digestion of many samples.
CAUTION: Cyanogen bromide is toxic and volatile, use it under the hood and dispose of wastes judiciously
1- Dilute virus (or protein) to 1 mg/ml with 0.1M Tris-HCl, pH 8, 3% SDS. Add dithiothreitol to 2mM and 2-hydroxyethyldisulfide (or cystamine) to 20mM. Heat 2 min at 100oC. (This step blocks SH groups.)
2- To a 30 uml aliquot, add 70 ul of formic acid containing 5 mg CNBr. To other aliquots add 70 ul formic acid with 0.7 mg CNBr and 70 ul with 0.1 mg CNBr.
3- Incubate samples 4 hr at room temperature; heat 5 min at 50oC in hood.
4- Neutralize 100 ul samples with 200 ul N-ethylmorpholine on ice.
5- Mix solution thoroughly with 5-10 volumes of reagent grade acetone, store 1 hr at -80oC or overnight at -20oC.
6- Centrifuge and discard supernatant. Dry pellet under vacuum and dissolve in about 100 ul of Laemmli cracking buffer. Boil 2 min; electrophorese 5 ml aliquot.
7- Digest with formic acid by a similar protocol. Omit CNBr; incubate an aliquot 2 hr at 55oC and another 2 hr at 75oC then go to step 4.
8- Electrophorese on Laemmli-SDS gels, 0.75 mm thick, with an 8-25% polyacrylamide gradient. Stain with silver by Morrissey's method [Anal. Biochem. 117, 307-310 (1981)] reducing incubation times 2-fold.
A- Do not reduce and alkylate prior to CNBr cleavage. Traces of iodide can oxidize and cleave trp. Purported CNBr cleavage of lysozyme at trp (Braunitzer and Aschauer, Z. Physiol. Chem. 356, 473) probably involves iodide. In our hands CNBr does not cleave trp-X bonds.
B- Begin by digesting standard proteins of known sequence. BSA is a convenient model that generates large, easily separated fragments. Partial digestion provides valuable information. For example, initial digestion products arise from a single cleavage, intermediates appear at low digestion levels and disappear at high levels, and final products should still be accumulating in the most extensive digestions. Logarithmically spaced dilutions of CNBr give the best range of digestion.
C- CNBr cleavage is inherently incomplete. If referees feel otherwise, adjust photographic exposures to satisfy their whims.
D- We've tried many solvents and find none superior to 70% formic acid.
E- Viruses can be dissolved directly in formic acid without isolating proteins.
F- Formic acid cleaves asp-pro bonds slowly at room temperature. Higher temperatures speed the reaction. TMV coat protein, with 2 asp-pro bonds, is a convenient model.
G- Formic acid or CNBr treatment produces multiple or fuzzy bands with some proteins. Prior reduction and alkylation does not improve patterns. Avoiding excess CNBr helps in some cases. It is now known that CNBr, like carbodiimides, can couple amino acids. This may explain some heterogeneity among CNBr products. Purer grades of formic acid do not improve cleavage; adding traces of formaldehyde to the formic acid does not hinder cleavage.
H- Partial cleavage patterns are more useful than "complete" digestion patterns. "Light" digestion, such that each met-X bond is about 1/10 cleaved, yields only "primary" digestion products. The number of bands larger than 1/2 the size of the native protein is a rough estimate of the number of methionines.
I- CNI under similar conditions specifically cleaves trp-X. Cleavage is less efficient than with CNBr and extensive reaction yields products which give fuzzy bands.
Useful recent articles
Kraft P, Mills J, Dratz E, Analytical Biochemistry 292, 76-86 (2001) Mass Spectrometric Analysis of Cyanogen Bromide Fragments of Integral Membrane Proteins at the Picomole Level: Application to Rhodopsin
Kaiser R, Metzka L, Analytical Biochemistry 266, 1-8 (1999) Enhancement of cyanogen bromide cleavage yields for methionyl-serine and methionyl-threonine peptide bonds