Profiling of Bacillus thuringiensis Biopesticides: A High-Resolution LC-MS/MS Approach for Subspecies Identification and Protein Quantification

The widespread use of Bacillus thuringiensis (Bt)-based biopesticides in organic farming presents challenges for limited data on insecticidal protein concentrations in treated samples and the environment after the application of Bt biopesticides. To address this, an LC-HR-MS/MS-PRM method for the identification and quantification of crystal proteins from Bt subspecies kurstaki, aizawai, and israelensis was developed. Marker peptides were selected based on untargeted analyses and validated for subspecies differentiation and quantification using stable-isotope-labeled (SIL) standards. The method demonstrated high sensitivity (ng/g range), repeatability precision, and selectivity across various matrices. Application to basil plants treated with Bt biopesticides confirmed accurate subspecies identification and protein quantification. This approach supports product quality control and environmental safety by providing qualitative and quantitative data on Bt toxins in treated samples depending on the applied Bt biopesticide. It offers a robust analytical tool that addresses key limitations of current approaches.