代谢重编程被认为是癌症的一个标志，伴随着代谢产物水平的改变可以对基因表达、细胞分化和肿瘤环境产生深远影响。然而，目前缺乏系统性的评估细胞色素代谢物谱定的灭活和提取程序。为此，本研究旨在建立一种没有泄漏的、不偏见的代谢物谱制备协议，用于HeLa癌细胞。我们评估了三种样品灭活液（液态氮、-40℃ 50% 甲醇、0.5℃ 正常盐水）和四种提取剂（-80℃ 80% 甲醇、0.5℃ 甲醇/氯仿/水[1:1:1 v/v/v]、0.5℃ 50% 乙腈、75℃ 70% 乙醇）共12种组合方法，用于粘附的HeLa癌细胞的全局代谢物分析制备。基于同位素稀释质谱法，使用气/液相色谱-质谱联用定量测定了43种代谢产物，包括糖磷酸、有机酸、氨基酸、腺苷酸和辅酶等参与中心碳代谢的代谢产物。结果表明，使用不同的样品制备程序用IDMS方法获得的细胞提取物中细胞内代谢产物的总量在21.51至295.33 nmol/百万细胞之间。在12种组合中，经过两次磷酸盐缓冲盐水(PBS)清洗、用液态氮灭活、再用50%乙腈提取的细胞被发现是获得细胞内代谢产物效率最高、样品制备过程中损失最小的最佳方法。此外，当这12种组合用于从三维肿瘤球体获得定量代谢组数据时，得到的结论也是一样的。此外，进行了一个案例研究，评估多柔比星(DOX)对粘附细胞和三维肿瘤球体的影响，使用定量代谢分析方法。使用有针对性的代谢组数据进行通路富集分析表明，DOX暴露会显著影响与AA代谢有关的通路，这可能与缓解氧化还原应激有关。令人惊讶的是，我们的数据表明，与二维（2D）细胞相比，三维（3D）细胞内谷氨酰胺水平的增加有助于在糖酵解受限后补充三羧酸循环(TCA)。综上所述，本研究为HeLa癌细胞在二维和三维细胞培养条件下的定量代谢组分析提供了一个良好的灭活和提取协议。基于此，定量的时间解析代谢物数据可以为生成代谢重编程的假说提供依据，揭示其在肿瘤发展和治疗中的重要作用。 Cary Press Limited拥有此处发布的所有权利。

Metabolic reprogramming has been coined as a hallmark of cancer, accompanied by which the alterations in metabolite levels have profound effects on gene expression, cellular differentiation, and the tumor environment. Yet a systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of tumor cells is currently lacking. To achieve this, this study is aimed at establishing an unbiased and leakage-free metabolome preparation protocol for HeLa carcinoma cell. We evaluated 12 combinations of quenching and extraction methods from three quenchers (liquid nitrogen, -40°C 50% methanol, 0.5°C normal saline) and four extractants (-80°C 80% methanol, 0.5°C methanol/chloroform/water [1:1:1 v/v/v], 0.5°C 50% acetonitrile, 75°C 70% ethanol) for global metabolite profiling of adherent HeLa carcinoma cells. Based on the isotope dilution mass spectrometry (IDMS) method, gas/liquid chromatography in tandem with mass spectrometry was used to quantitatively determine 43 metabolites including sugar phosphates, organic acids, amino acids (AAs), adenosine nucleotides, and coenzymes involved in central carbon metabolism. The results showed that the total amount of the intracellular metabolites in cell extracts obtained using different sample preparation procedures with the IDMS method ranged from 21.51 to 295.33 nmol per million cells. Among 12 combinations, cells that washed twice with phosphate buffered saline (PBS), quenched with liquid nitrogen, and then extracted with 50% acetonitrile were found to be the most optimal method to acquire intracellular metabolites with high efficiency of metabolic arrest and minimal loss during sample preparation. In addition, the same conclusion was drawn as these 12 combinations were applied to obtain quantitative metabolome data from three-dimensional (3D) tumor spheroids. Furthermore, a case study was carried out to evaluate the effect of doxorubicin (DOX) on both adherent cells and 3D tumor spheroids using quantitative metabolite profiling. Pathway enrichment analysis using targeted metabolomics data showed that DOX exposure would significantly affect AA metabolism-related pathways, which might be related to the mitigation of redox stress. Strikingly, our data suggested that compared to two-dimensional (2D) cells the increased intracellular glutamine level in 3D cells benefited replenishing the tricarboxylic acid (TCA) cycle when the glycolysis was limited after dosing with DOX. Taken together, this study provides a well-established quenching and extraction protocol for quantitative metabolome profiling of HeLa carcinoma cell under 2D and 3D cell culture conditions. Based on this, quantitative time-resolved metabolite data can serve to the generation of hypotheses on metabolic reprogramming to reveal its important role in tumor development and treatment.© 2023 Wiley-VCH GmbH.