13C isotopomer analysis of glutamate by tandem mass spectrometry
13C isotopomer analysis of glutamate by tandem mass spectrometry
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DOI：
10.1006/abio.2001.5457
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发表时间：
2002-01-15
影响因子：
2.9
通讯作者：
Malloy, CR
中科院分区：
文献类型：
--
作者：
Jeffrey, FMH;Roach, JS;Malloy, CR
串联质谱法允许将化合物从样品的其余部分分离并解离成较小的片段。我们在这里表明，谷氨酸质量同位素的碎片产生额外的质谱数据，显着提高了代谢通量的分析相比，全扫描质谱。为了验证该技术，将串联和全扫描质谱法与C-13 NMR沿着用于分析用三种底物混合物（5 mM葡萄糖加5 mM [2-C-13]乙酸盐、5 mM [1-C-13]葡萄糖加5 U/L胰岛素和5 mM葡萄糖加1 mM [3-C-13]丙酮酸盐）灌注的大鼠心脏的谷氨酸盐。串联质谱分析显示，富集底物分别贡献了98 +/- 2、53 +/- 2和84 +/-7%的乙酰辅酶A，而回补底物进入率为7 +/- 3、25 +/- 8和16 +/-8%。用C-13 NMR数据得到了类似的结果，而全扫描数据的值具有较高的误差。我们相信，这是第一次使用串联质谱法来确定使用C-13富集底物的途径通量。虽然通过NMR分析柠檬酸循环更简单（也更直观），但串联质谱法具有将高灵敏度与以前仅通过NMR获得的高信息产量相结合的潜力。(C)2001年，爱思唯尔科学。
Tandem mass spectrometry allows a compound to be isolated from the rest of the sample and dissociated into smaller fragments. We show here that fragmentation of glutamate mass isotopomers yields additional mass spectral data that significantly improve the analysis of metabolic fluxes compared to full-scan mass spectrometry. In order to validate the technique, tandem and full-scan mass spectrometry were used along with C-13 NMR to analyze glutamate from rat hearts perfused with three substrate mixtures (5 mM glucose plus 5 mM [2-C-13]acetate, 5 mM [1-C-13]glucose plus 5 U/L insulin, and 5 mM glucose plus 1 mM [3-C-13]pyruvate). Analysis by tandem mass spectrometry showed that the enriched substrate contributed 98 +/- 2, 53 +/- 2, and 84 +/- 7%, respectively, of acetyl-coenzyme A while the rate of anaplerotic substrate entry was 7 +/- 3, 25 +/- 8, and 16 +/- 8%. Similar results were obtained with C-13 NMR data, while values from full-scan data had higher error. We believe that this is the first use of tandem mass spectrometry to determine pathway flux using C-13-enriched substrates. Although analysis of the citric acid cycle by NMR is simpler (and more intuitive), tandem mass spectrometry has the potential to combine high sensitivity with the high information yield previously available only by NMR. (C) 2001 Elsevier Science.