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An automated fluorescence protein sequencer using 7‐methylthio‐4‐(2,1,3‐benzoxadiazolyl) isothiocyanate (MTBD‐NCS) as an Edman reagent
Author(s) -
Okiyama Natsuko,
Santa Tomofumi,
Toriba Akira,
Nakagomi Kazuya,
Imai Kazuhiro,
Hiranuma Hidenori,
Tanaka Hiroshi
Publication year - 2002
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.124
Subject(s) - chemistry , reagent , isothiocyanate , fluorescence , chromatography , edman degradation , fluorescein isothiocyanate , fluorescein , biochemistry , organic chemistry , peptide sequence , physics , quantum mechanics , gene
An automated fluorescence protein sequencer using 7‐methylthio‐5‐(2,1,3‐benzoxadiazolyl) isithiocyanate (MTBD‐NCS), a fluorescent Edman reagent, is developed by the modification of a commercial protein sequencer. The generated MTBD‐thiohydantoin amino acids fluoresced strongly, whereas the by‐products such as MTBD‐thiocarbamoyl amino acids and MTBD‐carbamoly amino acids did not fluoresce. A few interfering peaks were observed in the chromatogram and amino acid sequence was easily determined. The coupling and cyclization/cleavage reaction conditions and extraction conditions of generated MTBD‐thiazolinone amino acids were optimized using an autonalyzer. Finally, the sequence of a synthetic peptide (25 pmol), leucine‐enkephalin‐Thr‐amide, was determined and up to six residues were successively analyzed. Copyright © 2002 John Wiley & Sons, Ltd.