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首页> 美国卫生研究院文献>Journal of Visualized Experiments : JoVE >Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation
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Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

机译：高效液相色谱-流动闪烁法检测磷酸肌醇的细胞水平的放射性标记和定量

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摘要

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.
机译：磷脂酰肌醇（PtdInsPs）是必需的信号脂质，负责募集特定的效应子并赋予具有分子同一性和功能的细胞器。七个PtdInsP的分布和丰度各不相同，这些都受到特定激酶和磷酸酶的严格调控。 PtdInsP的丰度可以响应各种信号事件或调节机制的干扰而突然改变。要了解这些事件如何导致PtdInsP数量的变化及其产生的影响，重要的是在信号事件发生之前和之后或在控制与异常情况之间量化PtdInsP水平。但是，由于它们的丰度和相似度低，因此量化每个PtdInsP的相对量可能具有挑战性。本文介绍了一种通过用 3 H-肌醇合成代谢标记细胞的方法来定量PtdInsP水平的方法，该方法已整合到PtdInsPs中。然后使磷脂沉淀并脱酰基。进一步提取所得的可溶性 3 H-甘油-肌醇苷，通过高效液相色谱（HPLC）分离，并通过流动闪烁进行检测。详细描述了酵母样品的标记和处理，以及HPLC和流动闪烁器的仪器设置。尽管丢失了有关酰基链含量的结构信息，该方法还是灵敏的，可以进行优化以同时量化细胞中所有七个PtdInsP。

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