Summary
The alkylated-thiohydantoin method for C-terminal sequencing makes a significant improvement to the thiohydantoin method first described by Schlack and Kumpf. Prior to cleavage from the protein, the C-terminal thiohydantoin is alkylated, making it a better leaving group than the unmodified thiohydantoin. The C-terminal alkylated-thiohydantoin can be cleaved from the protein under conditions that simultaneously form the next thiohydantoin. Combining cleavage and thiohydantoin formation in one step eliminates the need for activating the C-terminal carboxyl group before every sequencing cycle and prevents detection of C-termini formed by random cleavage of peptide bonds in the protein during the sequencing chemistry. The alkylated-thiohydantoin method includes the presequencing modification of cysteine and lysine and the automated modification of aspartic and glutamic acids, serine and threonine. Modifying the reactive side-chain groups improves the ability to sequence through and detect these amino acids. The alkylated-thiohydantoin method can sequence through and detect 19 of the 20 genetically coded amino acids. Sequencing stops at proline residues.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsPreview
Unable to display preview. Download preview PDF.
References
Edman P (1950) Method for the determination of the amino acid sequence of peptides. Acta Chem Scand 4: 283–293
Schlack P, Kumpf W (1926) Cher eine neue Methode zur Ermittlung der Konstitution von Peptiden. Z Physiol Chem 154: 125–170
Inglis AS (1991) Chemical procedures for C-terminal sequencing of peptides and proteins. Anal Biochem 195: 183–196
Boyd VL, Bozzini M, Zon G, Noble RL, Mattaliano RJ (1992) Sequencing of peptides and proteins from the carboxy terminus. Anal Biochem 206: 344–352
Martinez A, Knappskog PM, Olafsdottir S, Doskeland AP Eiken HG, Svebak RM, Bozzini M, Apold J, Flatmark T (1995) Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme. Biochem J 306: 589–597
Lu KV, Rohde MF, Thomason AR, Kenney WC, Lu HS (1995) Mistranslation of a TGA termination codon as tryptophan in recombinant platelet-derived growth factor expressed in Escherichia coli. Biochem J 309: 411–417
Bozzini M, Zhao J, Yuan P-M, Ciolek D, Pan Y-C, Horton J, Marshak DR, Boyd VL (1995) Applications using an alkylation method for carboxy-terminal sequencing. In: J Crabb (ed) Techniques in protein chemistry VI. Academic Press, San Diego, 229–237
Boyd VL, Bozzini M, Guga PJ, DeFranco RJ, Yuan P-M (1992) Activation of the carboxy terminus of a peptide for carboxy-terminal sequencing. J Org Chem 60: 2581–2587
Buchanan GL (1988) The Dakin-West reaction. Chem Soc (London) Reviews 17: 91–109
Stark GR (1968) Sequential degradation of peptides from their carboxyl termini with ammonium thiocyanate and acetic anhydride. Biochemistry 7: 1796–1807
Brune DC (1992) Alkylation of cysteine with acrylamide for protein sequence analysis. Anal Biochem 207: 285–290
Blagbrough IS, Mackenzie NE, Ortiz C, Scott AI (1986) The condensation reaction between isocyanates and carboxylic acids. A practical synthesis of substituted amides and anilides. Tetrahedron Lett 27: 1251–1254
Kenner GW, Khorana HG, Stedman RJ (1953) Peptides. Part IV. Selective removal of the C-terminal residue as a thiohydantoin. The use of diphenylphosphoroisothiocyanatidate. Chem Soc J (London), 673–678
Bailey JM, Nikfarjam F, Shenoy NR, Shively JE (1992) Automated carboxy-terminal sequence analysis of peptides and proteins using diphenylphosphoroisothiocyanatidate. Protein Sci 1: 1622–1633
Anumula KR, Tang S (1995) Novel chemistry for sequencing of proteins from the carboxyl terminus yields a simple method. FASEB J9: A1477
Mo B, Li J, Liang S (1997) Chemical carboxy-terminal sequence analysis of peptides using acetyl isothiocyanate. Anal Biochem 252: 169–176
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Springer Basel AG
About this chapter
Cite this chapter
Dupont, D.R., Bozzini, M., Boyd, V.L. (2000). The alkylated thiohydantoin method for C-terminal sequence analysis. In: Jollès, P., Jörnvall, H. (eds) Proteomics in Functional Genomics. EXS, vol 88. Birkhäuser, Basel. https://doi.org/10.1007/978-3-0348-8458-7_8
Download citation
DOI: https://doi.org/10.1007/978-3-0348-8458-7_8
Publisher Name: Birkhäuser, Basel
Print ISBN: 978-3-0348-9576-7
Online ISBN: 978-3-0348-8458-7
eBook Packages: Springer Book Archive