A pseudotargeted metabolomics method for phosphatidyl amino acid analysis in chemotherapy-resistant cells and...

Phosphatidyl amino acids (p-AAs) are metabolites characterized by the phosphorylation of the hydroxyl, amino, carboxyl, and thiol groups of amino acids. Previous research has primarily focused on the phosphorylation sites within macromolecular proteins, with a particular emphasis on typical O-p-AAs. In this study, we established a prediction library of p-AAs based on existing knowledge. To improve detection rates of p-AAs in biological samples, we employed a chemical labeling-based LC-MS/MS method, utilizing p-[3,5-(dimethylamino)-2,4,6-triazine] benzene-1-sulfonyl piperazine (Tmt-PP) and its deuterated form (d12-Tmt-PP) as paired labeling reagents. A preliminary identification was performed by matching characteristic MS fragments with available standards. Additionally, strategies such as in vitro methods were implemented for further identification. The phosphatase treatment aids in identifying phosphate-modified metabolites by dephosphorylating them, while cell extract incubation helps determine if novel phosphorylated amino acids are generated in vivo. Ultimately, we identified 11 p-AAs, 6 of which are novel metabolites reported for the first time. A pseudotargeted metabolomics method covering 11 identified p-AAs was established and applied to investigate the differences between cisplatin-resistant non-small cell lung cancer (NSCLC) cells and their parental cells, as well as their derived exosomes. This approach enhances our understanding of the role of p-AAs in various health and disease conditions and contributes to the discovery of additional novel phosphatidyl metabolites.