LCMS/MS-Based Mining of the Peptaibiome: A Discovery of New Peptaibols

In this study, we employed liquid chromatography–tandem mass spectrometry (LC–MS/MS) to perform chemo-profiling on four fungal species: three Trichoderma species─two strains of Trichoderma asperellum (S3–3 and S3–1) and one Trichoderma longibrachiatum (FS-46)─and one species from the Tolypocladium genus, Tolypocladium inflatum (MTCC-3538). Our analysis identified 78 peptaibols featuring α-aminoisobutyric acid (Aib). Among these, we discovered both cyclic and linear Peptaibols and lipopeptaibols with Aib, ranging in putative molecular weights from 1033.4 to 1953.6 g/mol, corresponding to peptide lengths of 10–20 amino acids. In total, 78 peptaibols, including lipopeptaibols, were identified through LC-MS/MS. Of these, 43 were recognized as new peptaibols based on mass fragmentation patterns and sequence data from the MS/MS analysis. Our findings suggest that LC-MS/MS-based large-scale studies of the peptaibiome will enable the discovery of novel peptaibols and provide a valuable reference for understanding fragmentation patterns of peptaibols, cyclic peptaibols, and lipopeptaibols. Moreover, LC-MS/MS allows rapid identification of peptaibiotics at low concentrations directly in extracts, offering a potential quality control method for selecting beneficial Trichoderma strains for crop protection, while their fragmentation patterns and plausible sequences can guide synthetic design.