Liu Xiaojing, Ser Zheng, Cluntun Ahmad A, Mentch Samantha J, Locasale Jason W
Division of Nutritional Sciences, Cornell University.
Division of Nutritional Sciences, Cornell University; Field of Biochemistry, and Molecular Cell Biology, Cornell University.
J Vis Exp. 2014 May 27(87):51358. doi: 10.3791/51358.
Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously. Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.
代谢物谱分析在健康和疾病代谢研究中一直是一项宝贵的资产。然而，当前的平台存在不同的限制因素，如劳动密集型的样品制备、低检测限、慢扫描速度、针对每种代谢物的密集方法优化，以及无法在单个实验中同时测量带正电荷和带负电荷的离子。因此，一种新颖的代谢组学方案可以推动代谢组学研究。基于酰胺的亲水色谱法能够在无需任何化学衍生化的情况下进行极性代谢物分析。使用Q-Exactive（QE-MS）的高分辨率质谱仪改善了离子光学性能，提高了扫描速度（在分辨率为70,000时为256毫秒），并且具有进行正/负切换的能力。采用冷甲醇萃取策略，并将酰胺柱与QE-MS联用，能够同时对168种靶向极性代谢物和数千个其他特征进行可靠检测。数据处理使用市售软件高效进行，从质谱图中提取的未知特征可以在数据库中进行查询。
