Direct analysis of PI(3,4,5)P3 using liquid chromatography electrospray ionization tandem mass spectrometry
Direct analysis of PI(3,4,5)P3 using liquid chromatography electrospray ionization tandem mass spectrometry
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DOI：
10.1016/j.ab.2018.02.014
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发表时间：
2018-04-15
影响因子：
2.9
通讯作者：
Fischl, Anthony S.
中科院分区：
文献类型：
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作者：
Bui, Hai H.;Sanders, Phillip E.;Fischl, Anthony S.
磷脂酰肌醇（3，4，5）三磷酸（PIP 3）是一种具有生物活性的膜磷脂，对所有真核细胞的生长和存活至关重要。我们描述了一种直接测量PIP 3的新方法，并描述了磷脂酰肌醇二磷酸，PI（3，5）P-2，PI（3，4）P-2和PI（4，5）P-2的位置异构体的HPLC分离和测量。采用超高效液相色谱（UHPLC）-电喷雾串联质谱（LC-ESI-MS/MS）在负多反应监测（MRM）模式下在线进行质谱分析。通过C18反相色谱法，向样品和移动的相中加入离子配对试剂二异丙基乙醇胺（DiiPEA）和乙二胺四乙酸四钠盐二水合物（EDTA），完成PIP 3与PI、磷脂酰肌醇磷酸（PIP）和PIP 2的快速分离，总运行时间（包括平衡）为12分钟。这些色谱条件导致样品之间没有PIP、PIP 2和PIP 3的残留。为了验证新方法，将U87 MG癌细胞血清饥饿并用PDGF处理以在存在或不存在磷脂酰肌醇3-激酶（PI 3 K）抑制剂LY 294002的情况下刺激PIP 3生物合成。LC/MS法生成的结果与使用[P-33]磷酸盐放射性标记的U87 MG细胞和阴离子交换色谱分析（一种经充分验证的测量PIP 3的方法）生成的结果非常一致。为了证明新方法的有用性，我们使用基于U87 MG细胞的测定法生成了几种充分表征的PI 3 K小分子抑制剂的可重现的IC 50数据，并显示可以从其他癌细胞系中测量PIP 3。总之，我们的结果表明，这种新方法是灵敏的，可重复的，可用于直接测量PIP 3没有放射性标记或复杂的脂质衍生化。
Phosphatidylinositol (3,4,5) trisphosphate (PIP3) is a biologically active membrane phospholipid that is essential for the growth and survival of all eukaryotic cells. We describe a new method that directly measures PIP3 and describe the HPLC separation and measurement of the positional isomers of phosphatidylinositol bisphosphate, PI(3,5)P-2, PI(3,4)P-2 and PI(4,5)P-2. Mass spectrometric analyses were performed online using ultra-high performance liquid chromatography (UHPLC)-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in the negative multiple-reaction monitoring (MRM) modes. Rapid separation of PIP3 from PI, phosphatidylinositol phosphate (PIP) and PIP2 was accomplished by C18 reverse phase chromatography with the addition of the ion pairing reagents diisopropylethanolamine (DiiPEA) and ethylenediamine tetraacetic acid tetrasodium salt dihydrate (EDTA) to the samples and mobile phase with a total run time, including equilibration, of 12 min. Importantly, these chromatography conditions result in no carryover of PIP, PIP2, and PIP3 between samples. To validate the new method, U87MG cancer cells were serum starved and treated with PDGF to stimulate PIP3 biosynthesis in the presence or absence of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Results generated with the LC/MS method were in excellent agreement with results generated using [P-33] phosphate radiolabeled U87MG cells and anion exchange chromatography analysis, a well validated method for measuring PIP3. To demonstrate the usefulness of the new method, we generated reproducible IC50 data for several well-characterized PI3K small molecule inhibitors using a U87MG cell-based assay as well as showing PIP3 can be measured from additional cancer cell lines. Together, our results demonstrate this novel method is sensitive, reproducible and can be used to directly measure PIP3 without radiolabeling or complex lipid derivatization.