农药在作物保护中越来越多地组合使用，导致对各种生物体的毒性增强。尽管蛋白质加合物组学具有挑战性，但在蛋白质组学方法和质谱高通量技术的最新进展的推动下，它仍然是一种强大的生物分析工具，可用于检查环境暴露并将外源加合物描述为高精度的假定毒性生物标志物。本研究旨在预测吡虫啉和高效氯氟氰菊酯杀虫剂对人类神经细胞的潜在神经毒性作用。我们的方案首先包括 3D 体外开发源自人脑肿瘤的神经球，然后用农药混合物进行处理。此外，我们采用了一种自下而上的蛋白质组学方法，使用纳流超高效液相色谱与高分辨率质谱仪进行蛋白质加合物分析并预测改变位点。选择两种蛋白质，即钙钙调蛋白依赖性蛋白激酶-II (CaMK2) 和膜联蛋白-A1 (ANXA1)，作为具有原始作用的关键靶标。从头测序揭示了由于神经毒性，82-ANXA1 和 228-CaMK2 的活性位点中形成了几种加合物，这是通过亲电子前体结构增加的质量位移预测的。据我们所知，我们的研究首次采用基于蛋白质组学的方法来深入研究农药分子相互作用及其加合蛋白质的潜力，而蛋白质加合在神经毒性机制中发挥着至关重要的作用。

Pesticides are increasingly used in combinations in crop protection, resulting in enhanced toxicities for various organisms. Although protein adductomics is challenging, it remains a powerful bioanalytical tool to check environmental exposure and characterize xenobiotic adducts as putative toxicity biomarkers with high accuracy, facilitated by recent advances in proteomic methodologies and a mass spectrometry high-throughput technique. The present study aims to predict the potential neurotoxicity effect of imidacloprid and λ-cyhalothrin insecticides on human neural cells. Our protocol consisted first of 3D in vitro developing neurospheroids derived from human brain tumors and then treatment by pesticide mixture. Furthermore, we adopted a bottom-up proteomic-based approach using nanoflow ultraperformance liquid chromatography coupled with a high-resolution mass spectrometer for protein-adduct analysis with prediction of altered sites. Two proteins were selected, namely, calcium-calmodulin-dependent protein kinase-II (CaMK2) and annexin-A1 (ANXA1), as key targets endowed with primordial roles. De novo sequencing revealed several adduct formations in the active site of 82-ANXA1 and 228-CaMK2 as a result of neurotoxicity, predicted by the added mass shifts for the structure of electrophilic precursors. To the best of our knowledge, our study is the first to adopt a proteomic-based approach to investigate in depth pesticide molecular interactions and their potential to adduct proteins which play a crucial role in the neurotoxicity mechanism.