Analysis of intact phosphoinositides in biological samples
Analysis of intact phosphoinositides in biological samples
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DOI：
10.1194/jlr.d600004-jlr200
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发表时间：
2006-07-01
影响因子：
6.5
通讯作者：
Wakelam, Michael J. O.
中科院分区：
文献类型：
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作者：
Pettitt, Trevor R.;Dove, Stephen K.;Wakelam, Michael J. O.
现在很明显，每种已知的天然存在的聚磷酸肌醇，磷脂酰肌醇单磷酸，（PtdIns 3 P、PtdIns 4P、PtdIns 5 P）、磷脂酰肌醇二磷酸[PtdIns（3，4）P-2、PtdIns（3，5）P-2、PtdIns（4，5）P-2]和磷脂酰肌醇三磷酸[PtdIns（3，4，5）P-3]在调节许多细胞事件中具有不同的作用，包括细胞内信号传导、迁移和囊泡运输。传统的鉴定技术需要[P-32]无机磷酸盐或[H-3]肌醇放射性标记、酸化脂质提取、脱酰和离子交换头基分离，这些方法耗时且不适用于放射性标记不切实际的样品，从而极大地限制了这些脂质在许多生理相关系统中的研究。因此，我们已经开发了一种新的，高效的，缓冲柠檬酸提取方法，以尽量减少酸诱导的磷酸肌醇降解，连同高灵敏度的液相色谱-质谱（LC-MS）协议，使用乙腈-氯仿-甲醇-水-乙胺梯度与微孔硅胶柱，使所有磷酸肌醇样品中的鉴定和定量。液相色谱法足以分离PtdInsP（3）和PtdInsP（2）区域异构体;然而，PtdInsP区域异构体需要LC和MS 3诊断裂解的组合。使用这种方法分析人血小板和酵母样品中的磷酸肌醇的数据。
It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4) P-2, PtdIns(3,5) P-2, PtdIns(4,5) P-2], and phosphatidylinositol trisphosphate [PtdIns(3,4,5) P-3], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [P-32] inorganic phosphate or [H-3] inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP(3) and PtdInsP(2) regioisomers; however, the PtdInsP regioisomers require a combination of LC and diagnostic fragmentation to MS3. Data are presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples.