Abstract
A novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2-carboxyethyl)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed-phase HPLC, are subjected to cleavage of the peptide bonds on the N-terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI-MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, beta-elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions.
Full Text
The Full Text of this article is available as a PDF (844.4 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Brew K., Castellino F. J., Vanaman T. C., Hill R. L. The complete amino acid sequence of bovine alpha-lactalbumin. J Biol Chem. 1970 Sep 10;245(17):4570–4582. [PubMed] [Google Scholar]
- Degani Y., Patchornik A. Cyanylation of sulfhydryl groups by 2-nitro-5-thiocyanobenzoic acid. High-yield modification and cleavage of peptides at cysteine residues. Biochemistry. 1974 Jan 1;13(1):1–11. doi: 10.1021/bi00698a001. [DOI] [PubMed] [Google Scholar]
- Gray W. R. Disulfide structures of highly bridged peptides: a new strategy for analysis. Protein Sci. 1993 Oct;2(10):1732–1748. doi: 10.1002/pro.5560021017. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gray W. R. Echistatin disulfide bridges: selective reduction and linkage assignment. Protein Sci. 1993 Oct;2(10):1749–1755. doi: 10.1002/pro.5560021018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jacobson G. R., Schaffer M. H., Stark G. R., Vanaman T. C. Specific chemical cleavage in high yield at the amino peptide bonds of cysteine and cystine residues. J Biol Chem. 1973 Oct 10;248(19):6583–6591. [PubMed] [Google Scholar]
- Kuwajima K., Ikeguchi M., Sugawara T., Hiraoka Y., Sugai S. Kinetics of disulfide bond reduction in alpha-lactalbumin by dithiothreitol and molecular basis of superreactivity of the Cys6-Cys120 disulfide bond. Biochemistry. 1990 Sep 11;29(36):8240–8249. doi: 10.1021/bi00488a007. [DOI] [PubMed] [Google Scholar]
- Lu H. S., Gracy R. W. 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. 1981 Dec;212(2):347–359. doi: 10.1016/0003-9861(81)90375-1. [DOI] [PubMed] [Google Scholar]
- Nakagawa S., Tamakashi Y., Ishibashi Y., Kawase M., Taketomi S., Nishimura O., Fukuda T. Production of human PTH(1-34) via a recombinant DNA technique. Biochem Biophys Res Commun. 1994 May 16;200(3):1735–1741. doi: 10.1006/bbrc.1994.1653. [DOI] [PubMed] [Google Scholar]
- Papayannopoulos I. A., Biemann K. Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry. Protein Sci. 1992 Feb;1(2):278–288. doi: 10.1002/pro.5560010210. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reeve J. R., Jr, Pierce J. G. Disulfide bonds of glycoprotein hormones. Their selective reduction in the beta subunits of bovine lutropin and thyrotropin. Int J Pept Protein Res. 1981 Jul;18(1):79–87. doi: 10.1111/j.1399-3011.1981.tb02042.x. [DOI] [PubMed] [Google Scholar]
- Regnier F. E., Gooding K. M. High-performance liquid chromatography of proteins. Anal Biochem. 1980 Mar 15;103(1):1–25. doi: 10.1007/978-1-4615-6728-8_1. [DOI] [PubMed] [Google Scholar]
- SMYTH D. G., STEIN W. H., MOORE S. The sequence of amino acid residues in bovine pancreatic ribonuclease: revisions and confirmations. J Biol Chem. 1963 Jan;238:227–234. [PubMed] [Google Scholar]
- Siuzdak G. The emergence of mass spectrometry in biochemical research. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11290–11297. doi: 10.1073/pnas.91.24.11290. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith D. L., Zhou Z. R. Strategies for locating disulfide bonds in proteins. Methods Enzymol. 1990;193:374–389. doi: 10.1016/0076-6879(90)93428-n. [DOI] [PubMed] [Google Scholar]
- Stark G. R. Cleavage at cysteine after cyanylation. Methods Enzymol. 1977;47:129–132. doi: 10.1016/0076-6879(77)47015-0. [DOI] [PubMed] [Google Scholar]
- Takeda K., Ogawa K., Ohara M., Hamada S., Moriyama Y. Conformational changes of alpha-lactalbumin induced by the stepwise reduction of its disulfide bridges: the effect of the disulfide bridges on the structural stability of the protein in sodium dodecyl sulfate solution. J Protein Chem. 1995 Nov;14(8):679–684. doi: 10.1007/BF01886906. [DOI] [PubMed] [Google Scholar]
- Wu J., Gage D. A., Watson J. T. A strategy to locate cysteine residues in proteins by specific chemical cleavage followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Biochem. 1996 Mar 15;235(2):161–174. doi: 10.1006/abio.1996.0108. [DOI] [PubMed] [Google Scholar]
- Zhou Z. R., Smith D. L. Assignment of disulfide bonds in proteins by partial acid hydrolysis and mass spectrometry. J Protein Chem. 1990 Oct;9(5):523–532. doi: 10.1007/BF01025005. [DOI] [PubMed] [Google Scholar]