Optimized peptide extraction method for analysis of antimicrobial peptide Kn2-7/dKn2-7 stability in human serum by
Wen Chen1, Dickson Kirui1, Nancy J Millenbaugh1
1Craniofacial Health & Restorative Medicine, Naval Medical Research Unit San Antonio, 3650 Chambers Pass, Building 3610, Joint Base San Antonio-Fort Sam Houston, TX 78234, USA.
Aim:
To develop an extraction protocol and determine stability for antimicrobial peptide (AMP) Kn2-7 and its d-enantiomer dKn2-7 in human serum.
Materials & Methods:
We compared use of ethanol, acetonitrile, RapiGest SF Surfactant and 1% formic acid in ethanol for AMP recovery from serum prior to liquid chromatography-mass spectrometry quantification.
Results:
Precipitation of samples with 1% formic acid in ethanol caused the least amount of AMP loss during extraction from serum. Time-course experiments revealed dKn2-7 was significantly more stable than Kn2-7 in 25% serum, with 78.5% of dKn2-7 and only 1.0% of Kn2-7 remaining after 24 h at 37°C.
Conclusion:
The optimized method significantly increased peptide recovery and allowed more accurate and consistent quantification of Kn2-7 and dKn2-7 serum stability.
