Phospholipid-Coated Fe3O4 Nanoparticles Enable Rapid Screening of Putative Antimicrobial Peptides from Clanis bilineata tsingtauica Hemolymph

Background/Objectives: Antimicrobial peptides (AMPs) in insect hemolymph are promising alternatives to conventional antibiotics, but their rapid screening from complex biological matrices remains difficult. This study aimed to develop a simple and efficient method for the enrichment and preliminary identification of putative AMPs from the hemolymph ofClanis bilineata tsingtauicalarvae.Methods: Phospholipid-coated magnetic nanoparticles (Fe3O4@L) were prepared using membrane phospholipids identified fromEnterococcus faeciumby time-of-flight mass spectrometry. After nanoparticle characterization, enrichment conditions were optimized and the Fe3O4@L workflow was compared with a conventional ultrafiltration-electrophoresis-silver staining-mass spectrometry (UESM) method. Peptides were identified by de novo mass spectrometry, followed by sequence filtering, replicate screening, and in silico AMP prediction. Candidate peptides were chemically synthesized and evaluated for antibacterial activity, minimum inhibitory concentration (MIC), growth inhibition, morphological effects, and hemolytic activity.Results: Optimization of the Fe3O4@L method resulted in a 1.86-fold increase in peptide concentration in the enriched fraction. Compared with UESM, Fe3O4@L reduced the workflow from four steps to three and shortened processing time by 61.2%. De novo analysis generated 724 filtered peptides, of which 58 were reproducibly detected in at least two replicates, yielding five putative AMPs after activity prediction; no putative AMP was identified by UESM under the same conditions. Among the synthesized peptides, CBT-3 showed antibacterial activity againstE. faeciumandEscherichia coli, with MICs of 32 and 64 mg/L, respectively, and low hemolytic activity toward human erythrocytes.Conclusions: Fe3O4@L provides a simple and effective strategy for preliminary AMP screening from insect hemolymph, and CBT-3 represents a promising peptide candidate for further investigation.