| Sequence analysis of proteins and peptides is an important technique in protein chemistry. The chemical degradation method established by Edman in 1950 has been a routine approach for N-terminal sequencing and is widely employed by automated protein sequencer. The C-terminal sequence is as important as N-terminal sequence in protein structure, and sequencing from c-terminus appears more and more important. Not only sequence information from the c-terminus of proteins and peptides is of especial interest in the investigation of N-terminally blocked proteins and peptides, but also C-terminal sequence analysis can facilitate the production of more specific probes for gene cloning. Among numerous strategies for C-terminal protein sequencing (iso)thiocyanate method established by Schlack-Kumpf in 1926 appears to be a promising approach because of the similarity to the Edman degradation in mechanism. However, due to the poor reactive carboxyl group of C-terminus, it is difficult to derivatize peptides topeptidyl thiohydantoins for C-terminus, and the Schlack-Kumpf degradation based on thiohydantoin procedure has not yet been developed to such an efficient method as Edman degradation. But in recent developments, especially in derivatizing reagents, have demonstrated that this method appears very promising in the field of protein C-terminal sequence analysis in future. Furthermore, with the significant improvements of mass spectrometry technologies, which facilitate analysis of largebiological molecules. Application of mass spectrometrytechnique for C-terminal sequence determination is a kind of new strategy, and now many achievements have been made in recent years. Mass spectrometry technique will play more and more role in the field of sequence analysis.Standard amino acid thiohydantoins are required as reference standard for development of C-terminal protein sequencing based on the thiohydantoin procedure. All of 19 amino acid thiohydantoins were synthesized by using free amino acids as starting materials, acetic anhydride as the carboxyl group activating reagent and TMS-ITC as the derivatizing reagent. The proiine thiohydantoin was prepared by using ammonium thiocyanate (N114SCN) as the derivatizing reagent. Products were purified by reversed phase I1PLC and characterized by ultraviolet spectra.( Iso ) thiocyanate method is also called Schlack-Kumpf degradation .We spend much time on the derivatizing reagent which is extremely crucial to this method. Four derivatizing reagents that seemed theoretically practical have been introduced, after thoroughly invest igation of them, two of which are practical and can derivatizing the amino acids to their corresponding thiohydantoins: (1)tributylsilyl isothiocyanate (TBuS-ITC); (2)triphenylgermanium isothiocyanate (TPGe-ITC). The two practical derivatizing reagents can be conveniently synthesized and characterized by mass spectra. Schlack-Kumpf degradation generally involves: (1) Carboxyl activation: the carboxyl group is activated by acetic anhydride and then produce peptidyl oxazolinone; (2) Coupling reaction: the reaction of pept idyl oxazol inone wi th one of the above derivztizing reagentsproduces peptidyl isothiocyanate; (3) Cyclization reaction: Cyclization reaction automatically forms peptidyl thiohydantoin; (4) Cleavage reaction: at last specific hydrolysis of peptidyl thiohydantoin is continued, then the amino acid thiohydantoin and a shortened peptide are obtained. A decapeptide (NH2-NYQKDALGFL-COOH ) and a hexadecapeptide ( NH2-KAKESDAGFLMFVYLV-COOH )were synthesized as model peptides, then covalently attached to DITC glass beads for methodology study in TBuS-ITC and TPGe-ITC chemistry. Four C-terminal residues of the synthetic decapeptide have been identified. The initial yield is 53.1% and the repetitive yield is 57.6%.What's more, a series of reaction conditions including activating reagent volume, activating time and derivatizing temperature, time have been investigated using decapeptide as model peptide and TPGe-ITC... |
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