A clean-up technology for the simultaneous determination of lysophosphatidic acid and sphingosine-1-phosphate by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a phosphate-capture molecule, Phos-tag
A clean-up technology for the simultaneous determination of lysophosphatidic acid and sphingosine-1-phosphate by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a phosphate-capture molecule, Phos-tag
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DOI：
10.1002/rcm.4484
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发表时间：
2010-04-01
影响因子：
2
通讯作者：
Satouchi, Kiyoshi
中科院分区：
文献类型：
--
作者：
Morishige, Jun-ichi;Urikura, Mai;Satouchi, Kiyoshi
溶血磷脂酸（LPA）和鞘氨醇-1-磷酸（S1 P）是具有磷酸基团的生长因子样脂质。血液中这些介质脂质的浓度被认为是早期检测癌症或血管疾病的潜在生物标志物。在这里，我们报告了一种方法，用于同时测定LPA和S1 P使用Phos标签，锌复合物，特异性结合到磷酸单酯基团。虽然LPA和S1 P都是亲水性化合物，但我们发现当它们与Phos-tag形成复合物时，它们获得了疏水性。基于这一发现，我们开发了一种从生物样品中富集LPA和S1 P的方法。在由氯仿/甲醇/水（1：1：0.9，v/v/v）组成的两相溶剂系统中进行第一次分配以除去脂质。LPA和SW通过添加Phos-标签在第二分区处被特异性地提取为Phos-标签复合物。LPA和S1 P的Phos标签复合物可通过基质辅助激光解吸/电离飞行时间质谱法（MALDI-TOFMS）检测，并且可基于离子的相对强度使用17：0 LPA和C17 S1 P作为内标物进行定量。通过分析小牛血清、大鼠脑和肺中的这些介质脂质，验证了该方案。净化方案是快速的，既不需要薄层色谱法（TLC），也不需要液相色谱法（LC），并适用于血液和固体组织样品。我们相信，我们的协议将是有用的LPA和SIP在许多临床样品的常规分析。版权所有（C）2010约翰威利父子有限公司
Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are growth factor-like lipids having a phosphate group. The concentrations of these mediator lipids in blood are considered to be potential biomarkers for early detection of cancer or vascular diseases. Here, we report a method for simultaneous determination of LPA and S1P using Phos-tag, a zinc complex that specifically binds to a phosphate-monoester group. Although both LPA and S1P are hydrophilic compounds, we found that they acquire hydrophobic properties when they form complexes with Phos-tag. Based on this finding, we developed a method for the enrichment of LPA and S1P from biological samples. The first partition in a two-phase solvent system consisting of chloroform/methanol/water (1:1:0.9, v/v/v) is conducted for the removal of lipids. LPA and SW are specifically extracted as Phos-tag complexes at the second partition by adding Phos-tag. The Phos-tag complexes of LPA and S1P are detectable by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and quantifiable based on the relative intensities of ions using 17:0 LPA and C17 S1P as internal standards. The protocol was validated by analyses of these mediator lipids in calf serum, a rat brain and a lung. The clean-up protocol is rapid, requires neither thin-layer chromatography (TLC) nor liquid chromatography (LC), and is applicable to both blood and solid tissue samples. We believe that our protocol will be useful for a routine analysis of LPA and SIP in many clinical samples. Copyright (C) 2010 John Wiley & Sons, Ltd.