Bioprospecting of Antimicrobial Peptides (AMPs) in Cannabis sativa L. Hemp Seeds Using Mass Spectrometry

Abstract

Hemp seeds (Cannabis sativa L.) are widely recognized for their nutritional value, particularly their high protein content and abundance of vitamins and minerals. However, their potential as a source of bioactive compounds, such as antimicrobial peptides (AMPs), remains relatively unexplored. This study investigates the presence of AMPs in hemp seed cake using nanoLC-MS/MS and peptide prediction algorithms, with the goal of identifying AMP candidates for pharmaceutical or agricultural applications. Proteins were extracted from Yum A hemp seed cake using a modified Osborne fractionation method, focusing on the water-soluble fraction. Extracted proteins were trypsin/Lys-C digested, followed by solid-phase extraction (SPE) using a Sep-Pak C18 cartridge. Protein concentrations were quantified before and after digestion and SPE using BCA assays. The resulting peptide samples were analyzed by LC-MS/MS, and the data were processed using PEAKS software version 12. Identified sequences were screened for antimicrobial potential using the CAMPR3 and AMP Scanner_V2 prediction algorithms. CAMPR3 predicted 33 unique AMP candidates, while AMP Scanner_V2 identified 294 unique candidates. Comparative analysis showed that 18 peptides were predicted by both platforms, while 15 peptides were uniquely identified by CAMPR3 and 276 were uniquely identified by AMP Scanner_V2. The overlapping peptides suggest higher-confidence AMP candidates, while the non-overlapping peptides highlight variability between prediction algorithms. These findings support the potential of hemp seed-derived peptides as a novel source of antimicrobial agents for future biotechnological and agricultural applications.