A novel derivatization strategy for profiling phosphate ester/anhydride metabolic network and application on glioma rats using HILIC-MS/MS

Phosphate esters and anhydrides have great significance in the field of biochemical research and medical therapy. The genetic materials (DNA or RNA), most of the coenzymes, many intermediary metabolites, such as nucleotides and glycosyl phosphates in vivo are phosphodiesters, phosphoric acid or phosphates, respectively. It is important to monitor endogenous active phosphate metabolites for investigating many biological processes or drug mechanism. However, the detection and determination of those free active phosphate metabolites are challenged due to their unstable and easily hydrolyzed property and relatively low sensitivity, especially diphosphates and triphosphates. In the current study, we successfully developed a strategy by 3-aminomethyl pyridine (AMPy) derivatization coupled with hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) for simultaneous determination of multiple types of phosphate metabolites with good stability in 48 h and 29 to 126-fold improvement of the limit of detection (LOD). Based on the diagnostic fragment ions of different types of AMPy-derivatized phosphate metabolites, characteristic MRM ion pairs were successfully performed for global profiling of the phosphate metabolites in phosphate ester/anhydride metabolic network, including nucleotide/deoxynucleotide mono/di/triphosphates, glycosyl mono/diphosphates, and other key phosphates, such as 5-phosphoribosyl-1-pyrophosphate (PRPP), SAICARP and FAICARP in HPF, HUVEC and PBMCs cells without standards. The developed strategy greatly expanded the coverage of applying a single derivatization reaction to analyze active phosphate metabolites. Finally, the established method was performed to investigate the phosphate esters and anhydrides based on a glioma rat model. For the first time, phosphate metabolites were comprehensively characterized based on phosphate ester and anhydride metabolic network, covering nucleotide metabolism, glycolysis and pentose phosphate pathways, etc. The results demonstrated that the applicability of the method could be extended to a wider range of active phosphate compounds and could facilitate to related applications in the future studies.

Automated summary

A strategy based on AMPy derivatization coupled with HILIC-MS/MS was successfully developed for the simultaneous determination of multiple types of phosphate metabolites, significantly improving the stability and sensitivity of detection, and expanding the coverage of analyzing active phosphate metabolites.