Optimized peptide extraction method for analysis of antimicrobial peptide Kn2-7/dKn2-7 stability in human serum by LC-MS

Wen Chen; Dickson Kirui; Nancy J Millenbaugh

doi:10.2144/fsoa-2022-0013

Optimized peptide extraction method for analysis of antimicrobial peptide Kn2-7/dKn2-7 stability in human serum by LC-MS

Future Sci OA. 2022 Jul 20;8(6):FSO807. doi: 10.2144/fsoa-2022-0013. eCollection 2022 Jul.

Authors

Wen Chen¹, Dickson Kirui¹, Nancy J Millenbaugh¹

Affiliation

¹ Craniofacial Health & Restorative Medicine, Naval Medical Research Unit San Antonio, 3650 Chambers Pass, Building 3610, Joint Base San Antonio-Fort Sam Houston, TX 78234, USA.

Abstract

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.

Keywords: antimicrobial peptide; liquid chromatography; mass spectrometry; peptide extraction; serum stability.