A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity

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• 作者：Jung H. Suh ; Abeer Eltanawy ; Apoorva Rangan ; Julie D. Saba ; ^{jsaba@chori.org" class="auth_mail" title="E-mail the corresponding author}
• 关键词：HQ ; 2-hydrazinoquinoline ; S1P ; sphingosine-1-phosphate ; LC/MS ; liquid chromatography/mass spectrometry ; C17-S1P ; sphingosine-1-phosphate ; MRM ; multiple reaction monitoring ; CE ; collision energy ; CV ; cone votage ; DNPH ; 2 ; 4-dinitrophenylhydrazine ; DAIH ; 2-diphenylacetyl-1 ; 3-indandion-1-hydrazone ; SPL ; sphingosine phosphate lyase
• 刊名：Chemistry and Physics of Lipids
• 出版年：2016
• 出版时间：January 2016
• 年：2016
• 卷：194
• 期：Complete
• 页码：101-109
• 全文大小：1193 K

文摘

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.