Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland 20850, United States.
Anal Chem. 2012 Mar 20;84(6):2892-9. doi: 10.1021/ac203453t. Epub 2012 Mar 7.
The absolute quantitation of amine metabolites from mammalian cell samples was achieved by combining amine standards, isobaric tags, and capillary liquid chromatography (LC) tandem mass spectrometry (MS/MS). Our approach allowed 32 specific amines to be analyzed within a single chromatographic run, with the generation of the calibration curve and absolute quantitation of each analyte taking less than 900 ms. Using this strategy, we determined the amine response of human aortic endothelial cells (HAECs) from a glucose challenge. The observed changes of the absolute concentration of these metabolites implied eight enzymatic reactions may change efficiency upon glucose treatment. Five of these reactions have been previously reported as being up-regulated in diabetic conditions. The remaining three reactions were analyzed by measuring the expression of these enzymes, with 66% showing increases. Our data indicate that rapid determination of absolute quantitation is useful in determining novel pathway activation. Furthermore, even though we determined the absolute quantity of 32 metabolites here, the number of analytes that can be measured by this method is limited mainly by commercial availability of amine standards.
采用胺标准品、等重标记物和毛细管液相色谱（LC）-串联质谱（MS/MS），实现了哺乳动物细胞样品中胺代谢物的绝对定量。我们的方法允许在单个色谱运行中分析 32 种特定的胺，生成校准曲线和每个分析物的绝对定量时间少于 900 毫秒。使用该策略，我们测定了葡萄糖刺激下人主动脉内皮细胞（HAEC）的胺反应。这些代谢物绝对浓度的变化表明，在葡萄糖处理时，八种酶反应的效率可能发生变化。其中五个反应在糖尿病条件下被报道为上调。其余三个反应通过测量这些酶的表达进行分析，其中 66%显示增加。我们的数据表明，快速测定绝对定量在确定新的通路激活方面很有用。此外，尽管我们在这里确定了 32 种代谢物的绝对数量，但该方法可测量的分析物数量主要受胺标准品的商业可用性限制。
