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. 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). Lay abstract: The rise of multidrug resistant (MDR) infections is a growing concern. Antimicrobial peptides (AMPs) have been found to be a promising new adjunct in the development of improved antimicrobial treatments to address this rise in MDR infections. The challenge of detecting and quantifying AMPs in biological matrices is a major limitation in fully developing this novel approach. Our work is a major step forward in providing a protocol for researchers who study a wide range of AMPs for many applications, in addition to the study of novel treatments for MDR infections.