Sheng Ning, Zhao Hongyi, Chen Xiong, Wang Dongmei, Li Menglin, Wang Zhe, Zhang Jinlan, Jiang Jiandong
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, PR China.
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, PR China.
Talanta. 2021 Jun 1;228:122238. doi: 10.1016/j.talanta.2021.122238. Epub 2021 Feb 20.
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.
磷酸酯和酸酐在生化研究和医学治疗领域具有重要意义。遗传物质（DNA或RNA）、大多数辅酶、许多中间代谢产物，如体内的核苷酸和糖基磷酸酯分别是磷酸二酯、磷酸或磷酸盐。监测内源性活性磷酸代谢产物对于研究许多生物学过程或药物作用机制很重要。然而，由于这些游离活性磷酸代谢产物不稳定、易水解且灵敏度相对较低，尤其是二磷酸酯和三磷酸酯，其检测和测定面临挑战。在本研究中，我们成功开发了一种策略，通过3-氨甲基吡啶（AMPy）衍生化结合亲水相互作用液相色谱-串联质谱（HILIC-MS/MS），用于同时测定多种类型的磷酸代谢产物，这些代谢产物在48小时内具有良好的稳定性，检测限（LOD）提高了29至126倍。基于不同类型的AMPy衍生化磷酸代谢产物的诊断性碎片离子，成功建立了特征性多反应监测（MRM）离子对，用于在无标准品的情况下对磷酸酯/酸酐代谢网络中的磷酸代谢产物进行全局分析，包括核苷酸/脱氧核苷酸单/二/三磷酸、糖基单/二磷酸以及其他关键磷酸盐，如5-磷酸核糖-1-焦磷酸（PRPP）、HPF中的SAICARP和FAICARP、人脐静脉内皮细胞（HUVEC）和外周血单个核细胞（PBMC）中的FAICARP。所开发的策略极大地扩展了应用单一衍生化反应分析活性磷酸代谢产物的覆盖范围。最后，基于胶质瘤大鼠模型，采用所建立的方法对磷酸酯和酸酐进行了研究。首次基于磷酸酯和酸酐代谢网络对磷酸代谢产物进行了全面表征，涵盖核苷酸代谢、糖酵解和磷酸戊糖途径等。结果表明，该方法的适用性可扩展到更广泛的活性磷酸化合物，并有助于未来研究中的相关应用。
