AI-assisted mass spectrometry imaging with in situ image segmentation for subcellular metabolomics analysis

Subcellular metabolomics analysis is crucial for understanding intracellular heterogeneity and accurate drug-cell interactions. Unfortunately, the ultra-small size and complex microenvironment inside the cell pose a great challenge to achieving this goal. To address this challenge, we propose an artificial intelligence-assisted subcellular mass spectrometry imaging (AI-SMSI) strategy with in situ image segmentation. Based on the nanometer-resolution MSI technique, the protonated guanine and threonine ions were respectively employed as the nucleus and cytoplasmic markers to complete image segmentation at the subcellular level, avoiding mutual interference of signals from various compartments in the cell. With advanced AI models, the metabolites within the different regions could be further integrated and profiled. Through this method, we decrypted the distinct action mechanism of isomeric drugs, doxorubicin (DOX) and epirubicin (EPI), only with a stereochemical inversion at C-4'. Within the cytoplasmic region, fifteen specific metabolites were discovered as biomarkers for distinguishing the drug action difference between DOX and EPI. Moreover, we identified that the downregulations of glutamate and aspartate in the malate-aspartate shuttle pathway may contribute to the higher paratoxicity of DOX. Our current AI-SMSI approach has promising applications for subcellular metabolomics analysis and thus opens new opportunities to further explore drug-cell specific interactions for the long-term pursuit of precision medicine.