Liu Huazhen, Yu Keshun, Kachroo Pradeep
Department of Plant Pathology, University of Kentucky, Lexington, Kentucky, USA.
Bio Protoc. 2023 Apr 5;13(7):e4645. doi: 10.21769/BioProtoc.4645.
Glycerol-3-phosphate (G3P) is a conserved precursor of glycerolipids that also plays an important role in plant defense. Its levels and/or metabolism are also associated with many human disorders including insulin resistance, diabetes, obesity, and cancer, among others. In plants, G3P accumulates upon pathogen infection and is a critical component of systemic acquired resistance, which confers broad spectrum disease resistance against secondary infections. G3P also plays an important role in root-shoot-root signaling in soybean that regulates incompatible interactions with nitrogen-fixing bacteria. Thus, accurate quantification of G3P is key to drawing a valid conclusion regarding its role in diverse processes ranging from lipid biosynthesis to defense. G3P quantification is further compounded by its rapid degradation in extracts prepared at room temperature. Here, we describe a simplified procedure for accurate quantitative analysis of G3P from plant tissues. G3P was extracted along with the internal standard ribitol, derivatized with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analyzed by gas chromatography-coupled mass spectrometry using selective ion mode. This procedure is simple, economical, and efficient, and does not involve isotopic internal standards or multiple-step derivatizations.
甘油-3-磷酸（G3P）是甘油脂的一种保守前体，在植物防御中也发挥着重要作用。其水平和/或代谢还与许多人类疾病相关，包括胰岛素抵抗、糖尿病、肥胖症和癌症等。在植物中，G3P在病原体感染时积累，是系统获得性抗性的关键成分，可赋予对二次感染的广谱抗病性。G3P在大豆的根-茎-根信号传导中也起着重要作用，该信号传导调节与固氮细菌的不相容相互作用。因此，准确量化G3P是就其在从脂质生物合成到防御等各种过程中的作用得出有效结论的关键。G3P在室温下制备的提取物中会迅速降解，这使得G3P的量化更加复杂。在此，我们描述了一种从植物组织中准确定量分析G3P的简化方法。G3P与内标核糖醇一起提取，用N-甲基-N-（三甲基硅基）三氟乙酰胺（MSTFA）衍生化，并使用选择性离子模式通过气相色谱-质谱联用进行分析。该方法简单、经济且高效，不涉及同位素内标或多步衍生化。
