Characterization of sphingosine-1-phosphate lyase activity by electrospray ionization-liquid chromatography/tandem mass spectrometry quantitation of (2E)-hexadecenal.
Characterization of sphingosine-1-phosphate lyase activity by electrospray ionization-liquid chromatography/tandem mass spectrometry quantitation of (2E)-hexadecenal.
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DOI：
10.1016/j.ab.2010.08.026
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发表时间：
2011-01-01
影响因子：
2.9
通讯作者：
Natarajan V
中科院分区：
文献类型：
--
作者：
Berdyshev EV;Goya J;Gorshkova I;Prestwich GD;Byun HS;Bittman R;Natarajan V
鞘鞘醇-1-磷酸（S1P）是一种鞘脂信号分子，对细胞存活和增殖至关重要。S1P介导的信号在很大程度上是通过其生物合成和降解来控制的，S1P裂解酶（S1PL）是唯一已知的酶，它可以不可逆地将鞘碱-1-磷酸降解为磷酸乙醇胺和相应的脂肪醛。s1pl介导的S1P降解生成(2E)-hexadecenal，而hexadecanal是二氢鞘氨醇-1-磷酸（DHS1P）降解的产物。脂肪醛可以通过生物转化为脂肪酸和/或醇，这使得它们难以捉摸，并使脂肪醛定量的任务具有挑战性。我们开发了一种简单、高灵敏度、高通量的方法，通过液相色谱-电喷雾电离-串联质谱法定量(2E)-十六烯醛作为氨基脲衍生物。该方法用于体外测定S1PL活性，每次测定使用低至0.25µg微粒体蛋白的能力。该方法也适用于使用组织匀浆作为S1PL的来源。（2E）-十六烯醛在酶促反应过程中由于生物转化而消失的校正是必需的，特别是在较高的蛋白质浓度下。该方法证实FTY720为S1PL抑制剂，IC50为52.4µM。
Sphingosine-1-phosphate (S1P) is a sphingolipid signaling molecule crucial for cell survival and proliferation. S1P-mediated signaling is largely controlled through its biosynthesis and degradation, and S1P lyase (S1PL) is the only known enzyme, which irreversibly degrades sphingoid base-1-phosphates to phosphoethanolamine and the corresponding fatty aldehydes. S1PL-mediated degradation of S1P results in the formation of (2E)-hexadecenal, while hexadecanal is the product of dihydrosphingosine-1-phosphate (DHS1P) degradation. Fatty aldehydes can undergo biotransformation to fatty acids and/or alcohols, which makes them elusive and renders the task of fatty aldehyde quantitation challenging. We have developed a simple, highly sensitive, and high-throughput protocol for (2E)-hexadecenal quantitation as a semicarbazone derivative by liquid chromatography-electrospray ionization-tandem mass spectrometry. The approach was applied to determining S1PL activity in vitro, with the ability to use as low as 0.25 µg microsomal protein per assay. The method is also applicable to the use of total tissue homogenate as the source of S1PL. A correction for (2E)-hexadecenal disappearance due to its biotransformation during enzymatic reaction is required, especially at higher protein concentrations. The method was applied to confirm FTY720 as the inhibitor of S1PL with the IC50 of 52.4 µM.
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影响因子：
3.9
作者：
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通讯作者：
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影响因子：
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DOI：
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发表时间：
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期刊：
Advances in enzyme regulation
影响因子：
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作者：
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通讯作者：
Saba, Julie D.