Diazonium-based derivatization for enhanced detection of phosphorylated metabolites by LC-MS in cells

Phosphorylated small molecule metabolites play crucial roles in physiological processes such as glycogen metabolism and inflammation regulation. However, their high polarity, structural similarity, poor chromatographic separation, and weak mass spectrometric signals make their accurate quantification challenging, thereby hindering the study of related metabolic mechanisms and diseases. To address these challenges, we developed a novel derivatization reagent, DMQX (5-diazomethane quinoxaline), and combined it with liquid chromatography-mass spectrometry (LC-MS). This approach achieved baseline separation of five groups of isomers and enabled the quantification of 24 phosphorylated metabolites, providing comprehensive coverage of these metabolites in biological pathways. We applied this method to quantify 21 endogenous phosphorylated metabolites in HepG2 cells with and without vesicular stomatitis virus infection, demonstrating the potential of this analytical approach for advancing the study of metabolic mechanisms through quantitative analysis of phosphorylated metabolites in biological samples.