Subcellular compartmentation has been challenging in plant 13C-metabolic flux analysis. Indeed, plant cells are highly compartmented: they contain vacuoles and plastids in addition to the regular organelles found in other eukaryotes. The distinction of reactions between compartments is possible when metabolites are synthesized in a particular compartment or by a unique pathway. Sucrose is an example of such a metabolite: it is specifically produced in the cytosol from glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). Therefore, determining the 13C-labeling in the fructosyl and glucosyl moieties of sucrose directly informs about the labeling of cytosolic F6P and G6P, respectively. To date, the most commonly used method to monitor sucrose labeling is by nuclear magnetic resonance, which requires substantial amounts of biological sample. This study describes a new methodology that accurately measures the labeling in free sugars using liquid chromatography tandem mass spectrometry (LC-MS/MS). For this purpose, maize embryos were pulsed with [U-13C]-fructose, intracellular sugars were extracted, and their time-course labeling was analyzed by LC-MS/MS. Additionally, extracts were enzymatically treated with hexokinase to remove the soluble hexoses, and then invertase to cleave sucrose into fructose and glucose. Finally, the labeling in the glucosyl and fructosyl moieties of sucrose was determined by LC-MS/MS.

中文翻译：

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糖中13 C-标记的液相色谱串联质谱定量。

在植物13C代谢通量分析中，亚细胞区划一直具有挑战性。实际上，植物细胞是高度分隔的：除了在其他真核生物中发现的常规细胞器外，它们还包含液泡和质体。当代谢物在特定的区室中或通过独特的途径合成时，区室之间的反应区分开是可能的。蔗糖就是这种代谢物的一个例子：它是在胞液中专门由6-磷酸葡萄糖（G6P）和果糖6-磷酸（F6P）产生的。因此，确定蔗糖的果糖基和葡糖基部分中的13 C-标记分别直接通知了胞质F6P和G6P的标记。迄今为止，最常用的监测蔗糖标记的方法是通过核磁共振，这需要大量的生物样品。这项研究描述了一种使用液相色谱串联质谱法（LC-MS / MS）准确测量游离糖中标记的新方法。为此，用[U-13C]果糖对玉米胚进行脉冲处理，提取细胞内糖，并通过LC-MS / MS分析其时程标记。另外，将提取物用己糖激酶进行酶处理以除去可溶性己糖，然后用转化酶将蔗糖切割成果糖和葡萄糖。最后，通过LC-MS / MS确定蔗糖的葡糖基和果糖基部分中的标记。用LC-MS / MS分析其时程标记。另外，将提取物用己糖激酶酶处理以除去可溶性己糖，然后用转化酶将蔗糖切割成果糖和葡萄糖。最后，通过LC-MS / MS确定蔗糖的葡糖基和果糖基部分中的标记。并用LC-MS / MS分析了它们的时程标记。另外，将提取物用己糖激酶酶处理以除去可溶性己糖，然后用转化酶将蔗糖切割成果糖和葡萄糖。最后，通过LC-MS / MS确定蔗糖的葡糖基和果糖基部分中的标记。