Jorge Tiago F, António Carla
Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), Oeiras, Portugal.
Methods Mol Biol. 2018;1778:71-86. doi: 10.1007/978-1-4939-7819-9_6.
Phosphorylated carbohydrates are central metabolites involved in key plant metabolic pathways, such as glycolysis and central carbon metabolism. Such pathways influence plant growth, development, and stress responses to environmental changes, and ultimately, reflect the plant's energy status. The high polarity of these metabolites, the variety of isomeric structures (e.g., glucose-1-phosphate (G1P)/fructose-6-phosphate (F6P)/mannose-6-phosphate (M6P)/G6P, sucrose-6-phosphate (S6P)/T6P), and rapid metabolic turnover makes their analysis particularly challenging. In this chapter, we describe the use of a set of known phosphorylated carbohydrates to develop and validate a hydrophilic interaction chromatography (HILIC) triple quadrupole (QqQ) tandem mass spectrometry (MS/MS) method in the highly sensitive and selective multiple reaction monitoring (MRM) mode for the target analysis of G1P, F6P, M6P, G6P, S6P, T6P, and the sugar nucleotide uridine 5-diphospho-glucose (UDPG). We present detailed information regarding HILIC column chemistry and practical considerations when coupling it with a QqQ-MS system.
磷酸化碳水化合物是参与植物关键代谢途径的中心代谢物，如糖酵解和中心碳代谢。这些途径影响植物的生长、发育以及对环境变化的应激反应，最终反映植物的能量状态。这些代谢物具有高极性、多种异构体结构（如葡萄糖 -1- 磷酸（G1P）/果糖 -6- 磷酸（F6P）/甘露糖 -6- 磷酸（M6P）/葡萄糖 -6- 磷酸（G6P），蔗糖 -6- 磷酸（S6P）/海藻糖 -6- 磷酸（T6P））以及快速的代谢周转，这使得它们的分析极具挑战性。在本章中，我们描述了使用一组已知的磷酸化碳水化合物来开发和验证一种亲水相互作用色谱（HILIC）三重四极杆（QqQ）串联质谱（MS/MS）方法，该方法在高灵敏度和选择性的多反应监测（MRM）模式下用于对G1P、F6P、M6P、G6P、S6P、T6P以及糖核苷酸尿苷5'-二磷酸葡萄糖（UDPG）进行靶向分析。我们提供了有关HILIC柱化学的详细信息以及将其与QqQ-MS系统联用的实际注意事项。
