HK2 Metabolomics

HK2 cells were incubated with interferon gamma or vehicle for 24 hours. HK2 cells were washed twice with ice-cold PBS, drained, snapped-frozen in liquid nitrogen and stored at -80°C until analyses. After addition of an extraction solution made of 50% methanol, 30% acetonitrile, and 20% water 33 (1 mL/1.106 cells or 500 l for 20 l urine), the samples were vortexed for 5 min at 4°C, and then centrifuged at 16,000 g for 15 min at 4°C. The supernatants were collected and separated by liquid chromatography–mass spectrometry using SeQuant ZIC-pHilic column (Millipore). The aqueous mobile-phase solvent was 20 mM ammonium carbonate plus 0.1% ammonium hydroxide solution and the organic mobile phase was acetonitrile. The metabolites were separated over a linear gradient from 80% organic to 80% aqueous for 15 min. The column temperature was 50°C and the flow rate was 200 μl/min. The metabolites were detected across a mass range of 75-1,000 m/z using the Q-Exactive Plus mass spectrometer at a resolution of 35,000 (at 200 m/z) with electrospray ionization and polarity switching mode. Lock masses were used to insure mass accuracy below 5 ppm. The peak areas of different metabolites were determined using Thermo TraceFinder software using the exact mass of the singly charged ion and known retention time on the HPLC column. Our metabolomics analyses are focused on small polar compounds in central carbon metabolism. We applied an established and largely referenced method for sample extraction and LC-MS analyses using pHILIC HPLC column for polar metabolites separation 33. Notably, we used the same extraction solution for cells and urine samples. As a part of the routine analytical pipeline, we apply the recommendations of the metabolomics Quality Assurance and quality Control Consortium (mQACC). The routine quality controls include regular equipment maintenance (Thermo), the use of standard operating procedures for sample extraction, storage and analyses. General practices also include weekly test runs to assure system stability and quality of runs. Regarding the QCs in relation to this study, we used (1) pooled interstudy QC, (2) process and extraction blanks, (3) system stability blanks, (4) solvents blanks, (5) long-term reference standard inter-laboratory QC mix to ensure system stability and (6) the samples were blinded and loaded in randomized order. The analyses of pooled samples QC showed no significant difference in metabolites levels between QCs.将HK2细胞与干扰素γ (interferon gamma) 或溶剂对照 (vehicle) 孵育24小时。用冰预冷的磷酸盐缓冲液 (PBS) 洗涤HK2细胞两次，沥干后置于液氮中快速冷冻，并于-80℃保存直至后续分析。加入由50%甲醇、30%乙腈和20%水组成的提取液（文献编号33：1 mL/1×10^6细胞，或针对20 μL尿液样本使用500 μL提取液），样本于4℃下涡旋振荡5分钟，随后在4℃、16000×g条件下离心15分钟。收集上清液，采用搭载SeQuant ZIC-pHilic色谱柱（默克密理博，Millipore）的液相色谱-质谱联用 (LC-MS) 系统进行分离。水相流动相为20 mM碳酸铵加0.1%氢氧化铵溶液，有机相流动相为乙腈。代谢物采用80%有机相到80%水相的线性梯度进行15分钟的分离。色谱柱温度设置为50℃，流速为200 μL/min。使用Q-Exactive Plus质谱仪，以电喷雾电离 (electrospray ionization) 和极性切换模式，在200 m/z处分辨率为35000的条件下，于75~1000 m/z的质荷比范围内检测代谢物。采用锁定质量 (lock masses) 确保质量准确度低于5 ppm。采用赛默飞TraceFinder软件，依据单电荷离子的精确质量与高效液相色谱 (HPLC) 柱上的已知保留时间，确定不同代谢物的峰面积。本研究的代谢组学分析聚焦于中心碳代谢中的小型极性化合物。我们采用了已建立且被广泛引用的方法，即使用亲水相互作用液相色谱 (pHILIC) 柱分离极性代谢物，进行样本提取与LC-MS分析（文献编号33）。值得注意的是，本研究对细胞样本与尿液样本使用了相同的提取液。作为常规分析流程的一部分，我们遵循了代谢组学质量保证与质量控制联盟 (metabolomics Quality Assurance and quality Control Consortium, mQACC) 的建议。常规质量控制措施包括：定期对设备（赛默飞，Thermo）进行维护、采用样本提取、存储与分析的标准操作流程；常规操作还包括每周开展测试运行，以确保系统稳定性与分析质量。针对本研究的质控措施，我们采用了：(1) 组间混合质控样；(2) 过程空白与提取空白；(3) 系统稳定性空白；(4) 溶剂空白；(5) 长期参考标准品室间质控混合样，以确保系统稳定性；(6) 样本采用盲法设计并以随机顺序上样。混合样本质控分析结果显示，不同质控样之间的代谢物水平无显著差异。