FAB-MSと酸分解法を用いる蛋白質・ペプチドのカルボキシ末端配列分析法
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概要
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We have developed the following unique methods essentially for carboxyl (C)-terminal sequencing of proteins and peptides.The first and important observation was the successive truncation reaction of peptide with the use of 90% pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA) at 90°C for 4-24 h by FAB and ESI-MS. This observation indicated two informations, one of which was the reaction-intermediate to be the prediction of the oxazolones at respective carboxyl termini of the truncated fragments, and the other was internal bond specific cleavages of carboxyl side of aspartic acid residue (Asp-C) and amino side of serine residue (Ser-N) as the side reactions.The prediction of oxazolone promoted to use perfluorate anhydride. The extensive successive truncation was also carried out even at -20°C within 1 h by the use of the perfluorate anhydride in acetonitrile solution.The side reactions, Asp-C and Ser-N were further studied; 0.2% PFPA aqueous vapor at 60°C for 24 h selectively cleaved Asp-Pro bond (60°C, h) and the same acid vapor at 90°C for 4-8 h cleaved Asp-C. An aqueous 90% PFPA vapor at 25°C for 48 h made N-O peptidyl shift at Ser/Thr. This was reacted with a vapor of 20% acetic anhydride and 1% acetic acid in acetoritrile at 60°C for 1 h. The reaction acetylated the free NH2-group of the Ser/Thr and the shifted peptide fragments. And finally 20% dimethylaminoethanol (DMAE) aqueous solution at 60°C for 30 min cleaved the O-ester peptide fragment off from OH-group of Ser/Thr residue.These two specific cleavages can be used for protein-identification by the aid of “peptide mass databases” made from protein sequence database.The successive truncation accompanied by Asp-C or Ser-N cleavages with 90% PFPA at 90°C for 1, 2, 4, and 8 h yielded multi-C-terminal sequences of protein. The data of several residues at several sites in protein provided information for protein identification again as well as for general gene technology. In the last two methodologies TOF-MS and FAB-MS were efficiently employed.A step wise C-terminal sequence method has been developed from the successive truncation reactions, which composed of the following three reactions; (1) formation of oxazolone and acetylation of the N-terminus with a vapor of acetic anhydride with 10% acetic acid at 60°C for 1 h; (2) cleavage off the C-terminal amino acid from the oxazolone and esterification of the peptide, and liberation of the C-terminal amino acid, with a vapor of 5% PFPA in methanol (ethanol) solution at 5°C for 5 min; and 3) hydrolysis of the peptide ester with 20% DMAE aqueous solution at 60°C for 10 min. And the products is ready to the next step reaction.
- 日本質量分析学会の論文
- 1997-10-01
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