Kinetic isotope tracing of de novo proteogenic amino acids and glycerol in A549 cells using [13C6]glucose

In this experiment, we aimed to monitor the metabolic intermediates including de novo amino acids in A549 cells upon exposure to U-C13 glucose. We observed that A549 cells produce kinetically labelled lactate, glycerol, and several amino acids that were incorporated in protein synthesis and contributed to biomass production. A549 cells grown on U-C13 glucose were used to extract the culture filtrate, methanolic metabolite fractions, and acid hydrolysate of proteins. These extracted metabolites were derivatised and analysed using gas chromatography and mass spectrometer (GC-MS). The GC-MS data were baseline corrected using MetAlign and the accurate mass isotopomer distribution (MIDs) were extracted for all labelled analytes. The mass isotopomer abundances were measured using Isocor software (Millard et al., 2012).

在本项实验中，我们旨在监测A549细胞在接触U-C13葡萄糖后的代谢中间产物，包括从头合成的氨基酸。观察到A549细胞产生动力学标记的乳酸、甘油以及数种参与蛋白质合成并促进生物量生成的氨基酸。培养于U-C13葡萄糖上的A549细胞被用于提取培养滤液、甲醇代谢物分馏物和蛋白质的酸水解物。这些提取的代谢物经过衍生化，并利用气相色谱-质谱联用技术（GC-MS）进行分析。GC-MS数据经MetAlign校正基线后，对所有标记的分析物提取了精确质量同位素分布（MIDs）。同位素丰度测量采用Isocor软件（Millard等，2012年）完成。