Biotechnology of Horticultural Crops, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Liesel-Beckmann-Straße 1, 85354 Freising, Germany.
Chair of Plant Nutrition, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Emil-Ramann-Straße 2, 85350 Freising, Germany.
Molecules. 2018 Jun 8;23(6):1389. doi: 10.3390/molecules23061389.
Glutamic and aspartic acid fulfil numerous functions in organisms. They are proteinogenic amino acids, they function as neurotransmitters, and glutamic acid links the citrate cycle with amino acid metabolism. In addition, glutamic acid is a precursor for many bioactive molecules like γ-aminobutyric acid (GABA). In tomatoes, glutamic acid accumulates in ripening fruits. Here we present a simple and rapid method for quantification of glutamate and aspartate in tomatoes. A cleared extract is prepared and 2-aminoadipic acid added as internal standard. Subsequently, the amino acids are derivatised with 2,4-dinitro-1-fluorobenzene under alkaline conditions. The derivatives are separated by ultra-high performance liquid chromatography using a phenyl-hexyl column and 50 mM -methylmorpholine/acetate buffer pH 7.4 containing 12% acetonitrile as eluent and detected by UV absorption at 363 nm. The whole analysis time including separation and column equilibration takes less than 2.8 min with a flow rate of 1 mL/min and less than 1.6 min with a flow rate of 2 mL/min, making this method suitable for high-throughput applications. The method shows excellent reproducibility with intra- and inter-day SDs of approximately 4% for both aspartic and glutamic acid. Using this method we show that the glutamate/aspartate ratio changes significantly during fruit ripening.
谷氨酸和天冬氨酸在生物体中具有多种功能。它们是蛋白质氨基酸，作为神经递质发挥作用，谷氨酸将柠檬酸循环与氨基酸代谢联系起来。此外，谷氨酸是许多生物活性分子如γ-氨基丁酸（GABA）的前体。在番茄中，谷氨酸在成熟果实中积累。在这里，我们提出了一种简单快速的方法来定量番茄中的谷氨酸和天冬氨酸。制备澄清提取物并加入 2-氨基己二酸作为内标。随后，在碱性条件下用 2,4-二硝基-1-氟苯将氨基酸衍生化。衍生化产物在苯基-己基柱上通过超高效液相色谱法分离，以 50 mM -甲基吗啉/乙酸盐缓冲液 pH 7.4 （含 12%乙腈）作为洗脱液，在 363nm 处通过紫外吸收检测。整个分析时间（包括分离和柱平衡）在 1mL/min 的流速下不到 2.8 分钟，在 2mL/min 的流速下不到 1.6 分钟，使该方法适合高通量应用。该方法具有出色的重现性，日内和日间的谷氨酸和天冬氨酸标准偏差约为 4%。使用该方法，我们表明在果实成熟过程中，谷氨酸/天冬氨酸的比值发生了显著变化。
