Polar Anionic Metabolome Analysis by Nano-LC/MS with a Metal Chelating Agent
Polar Anionic Metabolome Analysis by Nano-LC/MS with a Metal Chelating Agent
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DOI：
10.1021/ac901269h
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发表时间：
2009-09-15
影响因子：
7.4
通讯作者：
Oda, Yoshiya
中科院分区：
文献类型：
--
作者：
Myint, Khin Than;Uehara, Taisuke;Oda, Yoshiya
我们已经开发了一种实用的方法，与使用纳米LC/MS系统的生物样品中的极性阴离子代谢物的综合分析。一个多胺键合的聚合物为基础的apHera NH 2柱，这是与碳酸铵缓冲液兼容，有效地保留阴离子极性代谢产物，如有机酸，硫酸盐和磷酸盐，但多重磷酸化或羧化的化合物显示出高度扭曲的色谱峰形状。我们发现，添加微量的金属螯合剂乙二胺四乙酸（EDTA）的样品溶液显着改善多电荷阴离子化合物的峰形，即使质谱显示没有痕量的加合物离子在EDTA的情况下。在全扫描模式下，典型极性阴离子代谢物的检测限为0.19至2.81 pmol。在优化了从样品制备到nano-LC/MS分析的所有程序后，我们将我们的方法应用于真实的生物样品：Hela细胞、小鼠脑、人脑脊液（CSF）和人血浆。结果表明，Hela细胞和脑组织中含有大量磷酸化代谢产物，而血浆和脑脊液（CSF）中主要含有有机酸。磷酸化化合物可能不会分泌到CSF/血浆中或可能在CSF/血浆中不稳定。最后，该方法被用来检查抗癌药物甲氨蝶呤（MIX），抑制嘌呤从头生物合成和胸苷生物合成的作用模式。除了预期的代谢物水平的变化，我们发现以前未报道的代谢物，可能是甲基化尿苷5 '-三磷酸（UTP），由MTX处理的Hela细胞产生。
We have developed a practical method for the comprehensive analysis of polar anionic metabolites in biological samples with the use of a nano-LC/MS system. A polyamine-bonded polymer-based apHera NH2 column, which is compatible with ammonium carbonate buffer, effectively retained anionic polar metabolites, such as organic acids, sulfates, and phosphates, but multiply phosphorylated or carboxylated compounds showed highly distorted peak shapes on chromatograms. We found that addition of a trace amount of the metal chelating reagent ethylenediaminetetraacetic acid (EDTA) to the sample solution dramatically improved peak shapes of multiply charged anionic compounds, even though the mass spectra showed no trace of adduct ions in the absence of EDTA. The detection limits of typical polar anionic metabolites in the full-scan mode were from 0.19 to 2.81 pmol. After optimization of all the procedures from sample preparation to nano-LC/MS analysis, we applied our method to real biological samples: Hela cells, mouse brain, human cerebrospinal fluid (CSF), and human plasma. Our results indicated that phosphorylated metabolites were abundant in Hela cells and brain, while plasma and cerebrospinal fluid (CSF) mostly contained organic acids. Phosphorylated compounds might not be secreted into CSF/plasma or might be unstable in CSF/plasma. Finally, the method was used to examine the mode of action of the anticancer drug methotrexate (MIX), which inhibits purine de novo biosynthesis and thymidine biosynthesis. In addition of the expected changes of metabolite levels, we found that a previously unreported metabolite, probably a methylated uridine 5'-triphosphate (UTP), was produced by MTX-treated Hela cells.