Investigation of Human Blood Plasma Sample Preparation for Performing Metabolomics Using Ultrahigh Performance Liquid Chromatography/Mass Spectrometry
Investigation of Human Blood Plasma Sample Preparation for Performing Metabolomics Using Ultrahigh Performance Liquid Chromatography/Mass Spectrometry
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DOI：
10.1021/ac8024569
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发表时间：
2009-05-01
影响因子：
7.4
通讯作者：
Guy, Philippe A.
中科院分区：
文献类型：
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作者：
Bruce, Stephen J.;Tavazzi, Isabelle;Guy, Philippe A.
在一项新的两步设计研究中，采用超高效液相色谱-飞行时间质谱仪(UPLC/TOFMS)联用技术，研究了用于代谢组谱分析的人血浆制备方法。四种不同的有机溶剂(乙腈、丙酮、甲醇和乙醇)被用来评估蛋白质沉淀法制备人血浆的效果。研究了样品制备的最佳条件，考虑了提取标记的数量、数据质量/重现性和柱寿命的延长。使用同位素标记的内标来监测数据。质量/再现性。凝胶电泳法还被用来测量“第一设计步骤”上清液中的蛋白质含量，以便评估在多次注射后将被注入/累积到柱上的蛋白质的量，这在全球代谢图谱研究中将是显而易见的。第二个设计步骤是在第一个步骤中获得的结果的基础上进行的，选择了四个最佳条件并进行了进一步的研究，考察了旋涡时间和温度对沉淀/提取的影响。两种选择的溶剂组成对全球代谢谱分析血浆的制备“最佳”：“甲醇/乙醇”(1：1，v/v)和“甲醇/乙腈/丙酮”(1：1：1，v/v/v)加入血浆(4：1比，400亩L总体积)。
The following study investigates the preparation of human blood plasma for metabolomic profiling analysis by ultra-high performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC/TOFMS) in a novel two-step design study. Four different organic solvents (acetonitrile, acetone, methanol, and ethanol) were used to assess human blood plasma preparation via protein precipitation. The optimal conditions for sample preparation were investigated, with consideration to the number of extracted markers, data quality/reproducibility, and column lifetime prolongation. Isotopically labeled internal standards were used to monitor data. quality/reproducibility. Gel electrophoresis was also used to measure the protein content in the supernatant of the "first design step" allowing assessment of the amount of protein that would be injected/accumulate onto the column after many injections that would be apparent in a global metabolic profiling study. The second design step followed on from the results obtained in step one, with four of the best conditions selected and further investigated, looking at the effects of vortex time and temperature on precipitation/extraction. Two choices of solvent compositions were found to be "optimal" for preparation of plasma for global metabolic profiling analysis; these were "'methanol/ethanol" (1:1, v/v) and "methanol/acetonitrile/acetone" (1: 1:1, v/v/v) added to plasma (4:1 ratio, 400 mu L total volume).