A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity.
A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity.
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DOI：
10.1016/j.chemphyslip.2015.09.006
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发表时间：
2016-01
影响因子：
3.4
通讯作者：
Saba JD
中科院分区：
文献类型：
--
作者：
Suh JH;Eltanawy A;Rangan A;Saba JD
描述了一种用于定量生物样品中磷酸鞘氨醇裂解酶活性的新技术。在该方法中，使用2-肼基喹啉将（2 E）-十六烯醛转化为相应的腙衍生物，以提高离子化效率和检测选择性。液相色谱分离和多反应监测-质谱的组合利用允许同时定量底物S1 P和产物（2 E）-十六烯醛。将（2 E）-d5-十六碳烯醛作为内标物加入可提高检测准确度和精密度。一个简单的一步衍生过程消除了进一步提取的需要。（2 E）-十六烯醛和鞘氨醇-1-磷酸的定量限分别为100和50 fmol。该检测试剂盒显示出宽的动态检测范围，可用于检测野生型细胞、SPL过表达细胞系和野生型小鼠组织中的低基础磷酸鞘氨醇裂解酶活性。与现有方法相比，该方法同时检测1-磷酸鞘氨醇和（2 E）-十六烯醛的能力大大提高了结果的准确性，并显示出优异的灵敏度和特异性，用于磷酸鞘氨醇裂解酶活性检测。
A new technique for quantifying sphingosine phosphate lyase activity in biological samples is described. In this procedure, 2-hydrazinoquinoline is used to convert (2E)-hexadecenal into the corresponding hydrazone derivative to improve ionization efficiency and selectivity of detection. Combined utilization of liquid chromatographic separation and multiple reaction monitoring-mass spectrometry allows for simultaneous quantification of the substrate S1P and product (2E)-hexadecenal. Incorporation of (2E)-d5-hexadecenal as an internal standard improves detection accuracy and precision. A simple one-step derivatization procedure eliminates the need for further extractions. Limits of quantification for (2E)-hexadecenal and sphingosine-1-phosphate are 100 and 50 fmol, respectively. The assay displays a wide dynamic detection range useful for detection of low basal sphingosine phosphate lyase activity in wild type cells, SPL-overexpressing cell lines, and wild type mouse tissues. Compared to current methods, the capacity for simultaneous detection of sphingosine-1-phosphate and (2E)-hexadecenal greatly improves the accuracy of results and shows excellent sensitivity and specificity for sphingosine phosphate lyase activity detection.