Document Detail

Selective determination of tryptophan-containing peptides through precolumn derivatization and liquid chromatography using intramolecular fluorescence resonance energy transfer detection.
MedLine Citation:
PMID:  17690426     Owner:  NLM     Status:  MEDLINE    
A selective and sensitive fluorometric determination method for native fluorescent peptides has been developed. This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the amino groups of tryptophan (Trp)-containing peptides. In this detection process, we monitored the FRET from the native fluorescent Trp moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 10 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated FRET most effectively. The OPA derivatives of the native fluorescent peptides emitted OPA fluorescence (445 nm) through an intramolecular FRET process when they were excited at the excitation maximum wavelength of the Trp-containing peptides (280 nm). The generation of FRET was confirmed through comparison with the analysis of a non-fluorescent peptide (C-reactive protein fragment (77 - 82)) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the Trp-containing peptides when performing LC on a reversed-phase column. The detection limits (signal-to-noise ratio = 3) for the Trp-containing peptides, at a 20-microL injection volume, were 41 - 180 fmol. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of the system that takes advantage of the conventional detection of OPA derivatives. Moreover, native non-fluorescent amines and peptides in the sample monitored at FRET detection are weaker than those of conventional fluorescence detection.
Makoto Yoshitake; Naoko Sejima; Hideyuki Yoshida; Kenichiro Todoroki; Hitoshi Nohta; Masatoshi Yamaguchi
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Analytical sciences : the international journal of the Japan Society for Analytical Chemistry     Volume:  23     ISSN:  0910-6340     ISO Abbreviation:  Anal Sci     Publication Date:  2007 Aug 
Date Detail:
Created Date:  2007-08-10     Completed Date:  2007-10-03     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8511078     Medline TA:  Anal Sci     Country:  Japan    
Other Details:
Languages:  eng     Pagination:  949-53     Citation Subset:  IM    
Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, Johnan, Fukuoka, Japan.
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MeSH Terms
Chromatography, Liquid / methods
Fluorescence Resonance Energy Transfer / methods*
Fluorescent Dyes / chemistry
Molecular Structure
Peptides / analysis*
Reproducibility of Results
Sensitivity and Specificity
Tryptophan / analysis*
Reg. No./Substance:
0/Fluorescent Dyes; 0/Peptides; 73-22-3/Tryptophan

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