Abstract
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).
Highlights
Ethanol was found to be the best solvent for antimicrobial peptide (AMP) extraction in biological matrices compared with other tested organic solvents and acids
No writing assistance was utilized in the production of this manuscript
MethodsMol. Biol. 1293, 123–135 (2015). 11. Gillette MA, Carr SA. Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nat. Methods 10(1), 28–34 (2013). 12. Cao L, Dai C, Li Z et al Antibacterial activity and mechanism of a scorpion venom peptide derivative in vitro and in vivo. PLoS ONE 7(7), e40135 (2012)
Summary
LC–MS analyses were performed using a two-component system composed of mobile phase A (10% ACN/0.1% FA in water) and mobile phase B (0.1% FA in 100% ACN) at a flow rate of 0.4 ml/min. Our optimized method was verified by applying the same approach to human serum samples spiked with 50 μg/ml scorpion AMPs Kn2-7 (FIKRIARLLRKIF) or its D-isomer (dKn2-7). A 100 μl of solution was transferred to 200 μl of 100% ethanol These samples were stored at 4◦C for 30 min and were subsequently centrifuged at 17,000 × g for 20 min. Extracted Kn2-7and dKn2-7 in spiked serum samples were quantified with two MRM transitions m/z 558.7/120.2 and m/z 558.7/129.1 by LC–MS analysis (Figure 2). The MS/MS settings were: collision energy 47.07 V and collision cell exit potential 17.07 V for m/z 558.7/120.2; and.
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