Identification of Antimicrobial Peptides from HAN-J6 Hemp Seeds Using Mass Spectrometry and De Novo Peptide Sequencing

Abstract

Antimicrobial peptides (AMPs) extracted from natural sources, especially plants, have been shown to effectively inhibit microbes and are therefore promising alternatives to conventional antimicrobial agents. Hemp seeds (Cannabis sativa L.) have a high protein concentration and may serve as an abundant source of bioactive peptides. However, little is known about the presence and diversity of AMPs derived from hemp seed proteins. This study aimed to extract and identify AMPs from HAN-J6 strain hemp seeds using de novo peptide sequencing with mass spectrometry, an approach that has not yet been used to characterize hemp seed-derived AMPs. HAN-J6 strain hemp seed cakes were first defatted and then subjected to alkaline extraction and acid precipitation to extract proteins for mass spectrometry analysis. The protein sample then underwent protein precipitation, trypsin/Lys-C digestion, and ZipTip desalting prior to mass spectrometry analysis. Liquid chromatography-mass spectrometry (LC-MS), performed with two initial injections and one injection that utilized an exclusion list revealed 3,985 peptide sequences through analysis with PEAKS 12 software. Two prediction algorithms, CAMP R3 and AMP Scanner_V2, were used to screen the sequences; CAMP R3 predicted 56 AMPs, while AMP Scanner_V2 predicted 260 AMPs from de novo sequence compiled list (ALC 80% and above). Twenty-seven peptides were predicted to be AMPs by both algorithms, making them the most likely antimicrobial candidates for future testing. Our results support that hemp is a previously underexplored source of AMPs and could be valuable for pathogen-fighting applications in fields such as agriculture and pharmaceuticals.