Urinary metabolomics TCMR

We conducted a targeted metabolite profiling of urines samples from 193 kidney transplant recipients, collected three months after transplantation at the time of protocol biopsy. This was done using Liquid Chromatography coupled to tandem Mass Spectrometry with SeQuant ZIC-pHilic columns, which permit the detection of complex hydrophilic and polar compounds. We proceeded to compare the urine metabolome of KTR according to the presence of T-cell mediated rejection according the Banff classification (n=20). Urines were centrifugated 5 min at 4000 g and the supernatant was 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.本研究针对193名肾移植受者（kidney transplant recipients, KTR）的尿液样本开展靶向代谢组学分析（targeted metabolite profiling），所有样本均采集于移植后3个月的方案活检时点。实验采用搭载SeQuant ZIC-pHilic亲水色谱柱的液相色谱-串联质谱（Liquid Chromatography coupled to tandem Mass Spectrometry）系统，该系统可实现复杂亲水性与极性化合物的检测。本研究依据班夫分类标准（Banff classification）判定T细胞介导排斥反应（T-cell mediated rejection）的有无，以此分组比较肾移植受者的尿液代谢组特征，其中存在该排斥反应的受试者共20例。 尿液样本经4000 g相对离心力离心5分钟后，取上清液储存于-80℃环境中直至后续分析。向样本中加入由50%甲醇、30%乙腈与20%纯水组成的萃取液（参考文献33；每1×10^6个细胞对应1 mL萃取液，20 μL尿液样本则对应500 μL萃取液），样本于4℃下涡旋振荡5分钟，随后以16000 g相对离心力在4℃条件下离心15分钟。收集上清液，采用搭载SeQuant ZIC-pHilic亲水色谱柱（默克密理博，Millipore）的液相色谱-质谱联用系统进行分离分析。水相流动相为20 mM碳酸铵溶液添加0.1%氢氧化铵，有机相流动相为乙腈。代谢物采用线性梯度洗脱程序分离，流动相从80%有机相逐步切换至80%水相，洗脱时长为15分钟。色谱柱柱温设置为50℃，流速为200 μL/min。采用Q-Exactive Plus质谱仪，以电喷雾电离结合极性切换模式，在75~1000 m/z的质量范围内进行代谢物检测，质谱分辨率设置为35000（在200 m/z条件下）。采用锁定质量数校正技术，确保质量精度控制在5 ppm以内。采用赛默飞世尔TraceFinder软件，依据单电荷离子的精确质量数与高效液相色谱柱上的已知保留时间，对不同代谢物的峰面积进行定量。本代谢组学分析聚焦于中枢碳代谢通路中的小型极性化合物。本研究采用已被广泛引用的成熟方法开展样本萃取与液相色谱-质谱分析，通过亲水相互作用高效液相色谱柱实现极性代谢物的分离（参考文献33）。值得注意的是，本研究对细胞样本与尿液样本采用了相同的萃取液配方。作为常规分析流程的一部分，本研究遵循代谢组学质量保证与质量控制联盟（metabolomics Quality Assurance and Quality Control Consortium, mQACC）的相关建议。常规质量控制措施包括定期开展仪器维护（赛默飞世尔，Thermo）、严格遵循样本萃取、储存与分析的标准操作流程。日常质控还包含每周开展系统测试运行，以确保系统稳定性与分析质量。针对本研究相关的质量控制，我们采用了以下质控策略：(1) 组间混合质控样本；(2) 流程空白与萃取空白；(3) 系统稳定性空白；(4) 溶剂空白；(5) 长期参考标准的室间质控混合液，以保障系统稳定性；(6) 所有样本均采用盲法处理并以随机顺序加载至仪器。混合质控样本的分析结果显示，不同质控样本间的代谢物水平无显著差异。