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One label-based fluorescence detection of a protease that cleaves the peptide bond between two specific amino acids

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Abstract

In order to detect a protease that cleaves the peptide bond between two specific amino acids via fluorescence, a synthetic peptide modified with two labels (a donor and an acceptor) for fluorescence resonance energy transfer (FRET) is commonly used. However, the preparation and optimization of a peptide for the sensitive and selective detection of a target protease are time-consuming. In this study, we report a simple method for fluorescence protease detection using a readily prepared, one label-based peptide. The fluorescence detection of botulinum neurotoxin type E light chain (BoNT/E-LC) is based on a two-step proteolytic cleavage that involves the use of BoNT/E-LC and an externally supplemented L-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves the specific peptide bond between arginine and isoleucine within C-terminally 7-amino-4-methylcoumarin (AMC)-labeled oligopeptide, leaving fragmented isoleucine–AMC. Subsequently, LAP cleaves the peptide bond between isoleucine and AMC, liberating fluorescent AMC. This method does not require two label-modified peptides. Capping the oligopeptide with the D-form of tyrosine does not result in better performance in terms of detection limit, although a higher concentration of LAP can be used. The detection limit for BoNT/E-LC in both phosphate-buffered saline and commercial bottled water is 2 ng mL−1 for an incubation period of 1 h. The fluorescence detection is selective for BoNT/E-LC among the four tested BoNTs. Fluorescence detection using one label can be readily applied to any type of proteases without using FRET.

Graphical abstract: One label-based fluorescence detection of a protease that cleaves the peptide bond between two specific amino acids

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Publication details

The article was received on 09 Feb 2017, accepted on 30 Apr 2017 and first published on 15 May 2017


Article type: Paper
DOI: 10.1039/C7AY00347A
Citation: Anal. Methods, 2017, Advance Article
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    One label-based fluorescence detection of a protease that cleaves the peptide bond between two specific amino acids

    E. Kim, S. Park and H. Yang, Anal. Methods, 2017, Advance Article , DOI: 10.1039/C7AY00347A

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