(13)C Metabolomics: NMR and IROA for Unknown Identification.
(13)C Metabolomics: NMR and IROA for Unknown Identification.
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DOI：
10.2174/2213235x04666160407212156
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发表时间：
2016-08
期刊：
影响因子：
--
通讯作者：
Edison AS
中科院分区：
文献类型：
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作者：
Clendinen CS;Stupp GS;Wang B;Garrett TJ;Edison AS
同位素比异常值分析（IROA）是一种非靶向代谢组学方法，其使用稳定同位素标记和LC-HRMS在不同实验条件下鉴定和相对定量生物样品中的代谢物。我们展示了一种方法，使用高灵敏度13 C NMR来识别未知的代谢产物分离分馏材料从IROA LC-HRMS实验。来自线虫秀丽隐杆线虫的IROA样品使用LC-HRMS使用5次重复注射并收集30秒级分进行分级。将其浓缩并通过13 C NMR分析。我们对C. elegans并收集2个相邻的LC级分。通过HRMS，一个含有至少2种已知代谢物，苯丙氨酸和肌苷，另一个含有色氨酸和未知特征，单一同位素质量为m/z 380.0742 [M+H]+。通过核磁共振，我们能够很容易地验证已知的化合物，然后我们确定了负责未知共振的自旋系统网络。在搜索BMRB数据库并比较LC-HRMS的分子式后，我们确定片段是修饰的邻氨基苯甲酸酯和磷酸修饰的葡萄糖。然后我们进行量子化学NMR化学位移计算以确定最可能的异构体，其为3 '-O-磷酸-β-D-吡喃葡萄糖基-邻氨基苯甲酸酯。这种化合物以前曾在同一种生物体中发现过，验证了我们的方法。我们能够通过将共振与NMR数据库匹配并使用化学位移计算来确定正确的异构体，从而对先前已知的代谢物进行去复制，并鉴定出数据库中没有的代谢物。该方法是有效的，并且可以用于使用用于IROA的相同材料来鉴定未知的感兴趣化合物。
Isotopic Ratio Outlier Analysis (IROA) is an untargeted metabolomics method that uses stable isotopic labeling and LC-HRMS for identification and relative quantification of metabolites in a biological sample under varying experimental conditions. We demonstrate a method using high-sensitivity 13C NMR to identify an unknown metabolite isolated from fractionated material from an IROA LC-HRMS experiment. IROA samples from the nematode Caenorhabditis elegans were fractionated using LC-HRMS using 5 repeated injections and collecting 30 sec fractions. These were concentrated and analyzed by 13C NMR. We isotopically labeled samples of C. elegans and collected 2 adjacent LC fractions. By HRMS, one contained at least 2 known metabolites, phenylalanine and inosine, and the other contained tryptophan and an unknown feature with a monoisotopic mass of m/z 380.0742 [M+H]+. With NMR, we were able to easily verify the known compounds, and we then identified the spin system networks responsible for the unknown resonances. After searching the BMRB database and comparing the molecular formula from LC-HRMS, we determined that the fragments were a modified anthranilate and a glucose modified by a phosphate. We then performed quantum chemical NMR chemical shift calculations to determine the most likely isomer, which was 3’-O-phospho-β-D-glucopyranosyl-anthranilate. This compound had previously been found in the same organism, validating our approach. We were able to dereplicate previously known metabolites and identify a metabolite that was not in databases by matching resonances to NMR databases and using chemical shift calculations to determine the correct isomer. This approach is efficient and can be used to identify unknown compounds of interest using the same material used for IROA.