Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone orbitrap mass spectrometer.
Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone orbitrap mass spectrometer.
复制标题
DOI：
10.1021/ac902837x
复制
发表时间：
2010-04-15
影响因子：
7.4
通讯作者：
Rabinowitz, Joshua D.
中科院分区：
文献类型：
--
作者：
Lu, Wenyun;Clasquin, Michelle F.;Melamud, Eugene;Amador-Noguez, Daniel;Caudy, Amy A.;Rabinowitz, Joshua D.
我们提出了一种液相色谱-质谱（LC-MS）方法，利用轨道阱的质量分辨能力，对已知和未预料到的代谢物进行敏感和特异性的平行测量，重点关注参与核心代谢的水溶性物质。反相液相色谱法，循环时间为25分钟，在C18柱上以三丁胺作为离子配对剂进行水-甲醇梯度。质谱部分包括在独立轨道rap（“Exactive”）上以1hz和100,000分辨率在负离子模式下从85 - 800 m/z进行全面扫描。80个代谢物标准品的中位检出限为5 ng/mL，线性范围通常≥100倍。对于标准品和酿酒酵母（贝克酵母）的细胞提取物，峰强度的平均运行间相对标准偏差为8%。在酵母中，我们检测到137种已知的化合物，它们的13c标记模式也可以被跟踪来探测代谢通量。在缺乏未知功能基因（YKL215C）的酵母中，我们观察到m/z 128.0351离子的积累，我们随后证实这是氧脯氨酸，从而将YKL215C注释为氧脯氨酸酶。这些例子证明了本方法对定量代谢组学、通量组学和发现代谢物谱分析的适用性。
We present a liquid chromatography-mass spectrometry (LC-MS) method that capitalizes on the mass-resolving power of the orbitrap to enable sensitive and specific measurement of known and unanticipated metabolites in parallel, with a focus on water soluble species involved in core metabolism. The reversed phase LC method, with a cycle time 25 min, involves a water-methanol gradient on a C18 column with tributylamine as the ion pairing agent. The MS portion involves full scans from 85 – 800 m/z at 1 Hz and 100,000 resolution in negative ion mode on a stand alone orbitrap (“Exactive”). The median limit of detection, across 80 metabolite standards, was 5 ng/mL with linear range typically ≥ 100-fold. For both standards and a cellular extract from Saccharomyces cerevisiae (Baker’s yeast), the median inter-run relative standard deviation in peak intensity was 8%. In yeast exact, we detected 137 known compounds, whose 13C-labeling patterns could also be tracked to probe metabolic flux. In yeast engineered to lack a gene of unknown function (YKL215C), we observed accumulation of an ion of m/z 128.0351, which we subsequently confirmed to be oxoproline, resulting in annotation of YKL215C as an oxoprolinase. These examples demonstrate the suitability of the present method for quantitative metabolomics, fluxomics, and discovery metabolite profiling.
登录
查看更多内容
影响因子：
3.1
作者：
Herebian, Diran;Zuehlke, Sebastian;Spiteller, Michael
通讯作者：
Spiteller, Michael
影响因子：
14.9
作者：
Kanehisa M;Araki M;Goto S;Hattori M;Hirakawa M;Itoh M;Katayama T;Kawashima S;Okuda S;Tokimatsu T;Yamanishi Y
通讯作者：
Yamanishi Y
影响因子：
3.2
作者：
GUTNICK, D;CALVO, JM;AMES, BN
通讯作者：
AMES, BN
DOI：
10.1016/j.jasms.2009.03.002
发表时间：
2009-08-01
影响因子：
3.2
作者：
Bateman, Kevin P.;Kellmann, Markus;Taylor, Lester
通讯作者：
Taylor, Lester
影响因子：
7.4
作者：
Buescher, Joerg Martin;Czernik, Dominika;Zamboni, Nicola
通讯作者：
Zamboni, Nicola