Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1992 Jan;1(1):68–80. doi: 10.1002/pro.5560010108

Automated carboxy-terminal sequence analysis of peptides.

J M Bailey 1, N R Shenoy 1, M Ronk 1, J E Shively 1
PMCID: PMC2142088  PMID: 1304884

Abstract

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on solution phase conditions for formation of the peptidylthiohydantoins with trimethylsilylisothiocyanate (TMS-ITC) and for hydrolysis of these peptidylthiohydantoins into an amino acid thiohydantoin derivative and a new shortened peptide capable of continued degradation (Bailey, J. M. & Shively, J. E., 1990, Biochemistry 29, 3145-3156). The current study is a continuation of this work and describes the construction of an instrument for automated C-terminal sequencing, the application of the thiocyanate chemistry to peptides covalently coupled to a novel polyethylene solid support (Shenoy, N. R., Bailey, J. M., & Shively, J. E., 1992, Protein Sci. I, 58-67), the use of sodium trimethylsilanolate as a novel reagent for the specific cleavage of the derivatized C-terminal amino acid, and the development of methodology to sequence through the difficult amino acid, aspartate. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene. The chemistry involves activation with acetic anhydride, derivatization with TMS-ITC, and cleavage of the derivatized C-terminal amino acid with sodium trimethylsilanolate. The thiohydantoin amino acid is identified by on-line high performance liquid chromatography using a Phenomenex Ultracarb 5 ODS(30) column and a triethylamine/phosphoric acid buffer system containing pentanesulfonic acid. The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids were found to sequence in high yield (90% or greater) except for asparagine and aspartate, which could be only partially removed, and proline, which was found not be capable of derivatization. In spite of these current limitations, the methodology should be a valuable new tool for the C-terminal sequence analysis of peptides.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bailey J. M., Shively J. E. Carboxy-terminal sequencing: formation and hydrolysis of C-terminal peptidylthiohydantoins. Biochemistry. 1990 Mar 27;29(12):3145–3156. doi: 10.1021/bi00464a035. [DOI] [PubMed] [Google Scholar]
  2. Calaycay J., Rusnak M., Shively J. E. Microsequence analysis of peptides and proteins. IX. An improved, compact, automated instrument. Anal Biochem. 1991 Jan;192(1):23–31. doi: 10.1016/0003-2697(91)90177-u. [DOI] [PubMed] [Google Scholar]
  3. Cromwell L. D., Stark G. R. Determination of the carboxyl termini of proteins with ammonium thiocyanate and acetic anhydride, with direct identification of the thiohydantoins. Biochemistry. 1969 Dec;8(12):4735–4740. doi: 10.1021/bi00840a012. [DOI] [PubMed] [Google Scholar]
  4. Dwulet F. E., Gurd F. R. Use of thiocyanic acid to form 2-thiohydantoins at the carboxyl terminus of proteins. Int J Pept Protein Res. 1979 Feb;13(2):122–129. doi: 10.1111/j.1399-3011.1979.tb01859.x. [DOI] [PubMed] [Google Scholar]
  5. Hawke D. H., Harris D. C., Shively J. E. Microsequence analysis of peptides and proteins. V. Design and performance of a novel gas-liquid-solid phase instrument. Anal Biochem. 1985 Jun;147(2):315–330. doi: 10.1016/0003-2697(85)90278-7. [DOI] [PubMed] [Google Scholar]
  6. Hawke D. H., Lahm H. W., Shively J. E., Todd C. W. Microsequence analysis of peptides and proteins: trimethylsilylisothiocyanate as a reagent for COOH-terminal sequence analysis. Anal Biochem. 1987 Nov 1;166(2):298–307. doi: 10.1016/0003-2697(87)90578-1. [DOI] [PubMed] [Google Scholar]
  7. Holcomb G. N., James S. A., Ward D. N. A critical evaluation of the selective tritiation method of determining C-terminal amino acids and its application to luteinizing hormone. Biochemistry. 1968 Apr;7(4):1291–1296. doi: 10.1021/bi00844a007. [DOI] [PubMed] [Google Scholar]
  8. Meuth J. L., Harris D. E., Dwulet F. E., Crowl-Powers M. L., Gurd F. R. Stepwise sequence determination from the carboxyl terminus of peptides. Biochemistry. 1982 Aug 3;21(16):3750–3757. doi: 10.1021/bi00259a005. [DOI] [PubMed] [Google Scholar]
  9. Rangarajan M., Darbre A. Studies on sequencing of peptides from the carboxyl terminus by using the thiocyanate method. Biochem J. 1976 Aug 1;157(2):307–316. doi: 10.1042/bj1570307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Shenoy N. R., Bailey J. M., Shively J. E. Carboxylic acid-modified polyethylene: a novel support for the covalent immobilization of polypeptides for C-terminal sequencing. Protein Sci. 1992 Jan;1(1):58–67. doi: 10.1002/pro.5560010107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Shively J. E., Miller P., Ronk M. Microsequence analysis of peptides and proteins. VI. A continuous flow reactor for sample concentration and sequence analysis. Anal Biochem. 1987 Jun;163(2):517–529. doi: 10.1016/0003-2697(87)90257-0. [DOI] [PubMed] [Google Scholar]
  12. Stark G. R. Sequential degradation of peptides from their carboxyl termini with ammonium thiocyanate and acetic anhydride. Biochemistry. 1968 May;7(5):1796–1807. doi: 10.1021/bi00845a026. [DOI] [PubMed] [Google Scholar]
  13. Williams M. J., Kassell B. A solid phase method for peptide sequencing from the carboxyl terminus. FEBS Lett. 1975 Jul 1;54(3):353–357. doi: 10.1016/0014-5793(75)80938-0. [DOI] [PubMed] [Google Scholar]
  14. Yamashita S. Sequential degradation of polypeptides from the carboxyl ends. I. Specific cleavage of the carboxyl-end peptide bonds. Biochim Biophys Acta. 1971 Feb 16;229(2):301–309. doi: 10.1016/0005-2795(71)90189-9. [DOI] [PubMed] [Google Scholar]

Articles from Protein science : a publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES