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Optimization of a peptide extraction and LC–MS protocol for quantitative analysis of antimicrobial peptides

We optimized a peptide extraction and LC–MS protocol for identification and quantification of antimicrobial peptides (AMPs) in biological samples. Amphipathic AMPs were extracted with various concentrations of ethanol, methanol, acetonitrile, formic acid, acetic acid or trichloroacetic acid in water...

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Detalles Bibliográficos
Autores principales: Chen, Wen, Hwang, Yoon Y, Gleaton, Jeremy W, Titus, James K, Hamlin, Nicholas J
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Future Science Ltd 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331757/
https://www.ncbi.nlm.nih.gov/pubmed/30652017
http://dx.doi.org/10.4155/fsoa-2018-0073
Descripción
Sumario:We optimized a peptide extraction and LC–MS protocol for identification and quantification of antimicrobial peptides (AMPs) in biological samples. Amphipathic AMPs were extracted with various concentrations of ethanol, methanol, acetonitrile, formic acid, acetic acid or trichloroacetic acid in water. Yields were significantly greater for extraction with 66.7% ethanol than other extraction methods. Liquid chromatography was accomplished on a C18 column with a linear gradient of acetonitrile–formic acid, and mass spectrometry detection was performed in the positive electrospray multiple reaction monitoring mode by monitoring the transitions at m/z 385.2/239.2 and m/z 385.2/112.0 (AMP 1018), m/z 418.1/104.1 and m/z 418.1/175.1 (Methionine-1018). This method was shown to be reliable and efficient for the identification and quantification of scorpion AMPs Kn2-7 and its D-isomer dKn2-7 in human serum samples by monitoring the transitions at m/z 558.7/120.2 and m/z 558.7/129.1 (Kn2-7/dKn2-7).