Selective Determination of Tryptophan-containing Peptides through Precolumn Derivatization and Liquid Chromatography Using Intramolecular Fluorescence Resonance Energy Transfer Detection
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- YOSHITAKE Makoto
- Faculty of Pharmaceutical Sciences, Fukuoka University
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- SEJIMA Naoko
- Faculty of Pharmaceutical Sciences, Fukuoka University
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- YOSHIDA Hideyuki
- Faculty of Pharmaceutical Sciences, Fukuoka University
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- TODOROKI Kenichiro
- Faculty of Pharmaceutical Sciences, Fukuoka University
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- NOHTA Hitoshi
- Faculty of Pharmaceutical Sciences, Fukuoka University
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- YAMAGUCHI Masatoshi
- Faculty of Pharmaceutical Sciences, Fukuoka University
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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-µL 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.
収録刊行物
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- Analytical Sciences
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Analytical Sciences 23 (8), 949-953, 2007
社団法人 日本分析化学会
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詳細情報 詳細情報について
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- CRID
- 1390001204258320000
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- NII論文ID
- 130004441517
- 10019747525
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- NII書誌ID
- AA10500785
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- ISSN
- 13482246
- 09106340
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- NDL書誌ID
- 8847840
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- 本文言語コード
- en
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- データソース種別
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- JaLC
- NDL
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