GC-MS analysis of 13C metabolic labeling of HeLa kyoto cells expressing DadA in answer to intramitochondrial pyruvate influx by Grubraw

We analysed 13C metabolic inclusion into aminoacids, lactate, and fatty acids in HeLa cell lines stably expressing Pseudomonas aeruginosa DadA (FAD-dependent D-amino acid dehydrogenase) gene with mitochondrial targeting (named Grubraw) after the addition of 12C D-alanine comparing to no D-alanine. We used HeLa with functional DadA and mutated DadA for control. In cells expressing functionally active DadA, D-alanine generates additional influx of intra-mitochondrial pyrovate. If cells grows on labelled carbone source and unlabeled D-alanine is used, this upcoming pyruvate shifts original mass-isotopologue distribution that can be registered by GC-MS.

Our cells were cultured for in a medium with labeled glucose or glutamine. To obtain current data at 90 min timescale, we changed rich growth medium with labelled glucose to pure phosphate medium containing labeled glucose as only carbon substrate. In 24h timescale, we added labelled glucose or glutamine into rich medimum and change it to fresh one 24h before the extraction.

We publish original CDF data from Shimadzu TQ8040 mass-spectrometer. In “G” folders we put experiments with glucose as the only carbon source. In “GQ” folders, there are measurements for glucose+glutamine experiments. G/Q in folder name indicates the experiment with labelled glucose or glutamine correspondingly (the other source is unlabelled). “90m”/”24h” - indicate the treatment scheme (see next section). “C12”/”C13” in folder name indicates using of isotopically labeled carbon source or non-labeled one. All files are named in a similar way. “M”/“D” in file name indicate using a cell line with mutated (M) or functional (D) DadA. “+”/“-” indicates the addition of D-alanine. First number is a biological replicate, second number - technical replicate.

我们分析了稳定表达带有线粒体靶向序列的铜绿假单胞菌DadA（FAD依赖性D-氨基酸脱氢酶）基因（命名为Grubraw）的HeLa细胞系中，13C代谢物掺入氨基酸、乳酸和脂肪酸的情况，对比了添加12C-D-丙氨酸与未添加D-丙氨酸的组别。同时以具有功能性DadA和突变型DadA的HeLa细胞作为对照。在功能活性DadA表达的细胞中，D-丙氨酸会产生线粒体内丙酮酸的额外流入途径。若细胞在标记碳源中培养，同时使用未标记的D-丙氨酸，此时新增的丙酮酸会改变原本的质量同位素分布，该变化可通过气相色谱-质谱联用（GC-MS）进行检测。

我们的细胞在标记葡萄糖或谷氨酰胺的培养基中培养。为获取90分钟时间尺度的实验数据，我们将富含营养的标记葡萄糖培养基更换为仅以标记葡萄糖作为唯一碳底物的纯磷酸盐培养基。对于24小时时间尺度的实验，我们在完全培养基中添加标记葡萄糖或谷氨酰胺，并在萃取前24小时更换为新鲜培养基。

我们公开了来自岛津TQ8040质谱仪的原始CDF数据。在"G"文件夹中存放了以葡萄糖作为唯一碳源的实验数据；在"GQ"文件夹中，存放了葡萄糖+谷氨酰胺联合培养的实验测量数据。文件夹名称中的"G"和"Q"分别对应实验使用标记葡萄糖或标记谷氨酰胺（另一碳源为未标记状态）。"90m"/"24h"代表实验处理方案（详见下一部分）。文件夹名称中的"C12"/"C13"分别代表使用同位素未标记碳源或同位素标记碳源。所有文件的命名规则均类似。文件名中的"M"/"D"分别代表使用携带突变型（M）或功能性（D）DadA的细胞系；"+"/"-"代表是否添加D-丙氨酸。第一个数字为生物学重复编号，第二个数字为技术重复编号。