Detection of choline and phosphatidic acid (PA) catalyzed by Phospholipase D (PLD) using MALDI-QIT-TOF/MS with 9-aminoacridine matrix
Detection of choline and phosphatidic acid (PA) catalyzed by Phospholipase D (PLD) using MALDI-QIT-TOF/MS with 9-aminoacridine matrix
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使用 MALDI-QIT-TOF/MS 和 9-氨基吖啶基质检测磷脂酶 D (PLD) 催化的胆碱和磷脂酸 (PA)
DOI：
10.1080/09168451.2014.910102
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Fukamizu A
中科院分区：
文献类型：
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作者：
Park KE;Kim JD;Nagashima Y;Kako K;Daitoku H;Matsui M;Park GG;Fukamizu A
磷脂酶D（PLD）催化磷脂酰胆碱（PC）的水解，导致胆碱和磷脂酸（PA）的产生，磷脂酰胆碱（PC）是质膜中最丰富的磷脂。胆碱是神经递质乙酰胆碱的前体，而PA作为具有多种生物学功能的细胞内脂质介质发挥作用。为了评估PLD的体外活性，PLD衍生的胆碱通常用放射性或非放射性方法进行分析。本研究建立了以9-氨基吖啶为基体的MALDI-QIT-TOF/MS同时测定胆碱和PA的新方法。标准校准曲线表明，胆碱和PA可以检测线性范围从0.05和1 pmol，分别。重要的是，该方法能够同时检测胆碱和PA作为PLD 2蛋白水解PC的反应产物。因此，我们简单而直接的方法将是有用的，以表征PLD的酶性质，从而提供洞察PLD激活的机制。
Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), the most abundant phospholipids of plasma membrane, resulting in the production of choline and phosphatidic acid (PA). Choline is a precursor of the neurotransmitter acetylcholine, whereas PA functions as an intracellular lipid mediator of diverse biological functions. For assessing PLD activityin vitro, PLD-derived choline has been often analyzed with radioactive or non-radioactive methods. In this study, we have developed a new method for detecting choline and PA with MALDI-QIT-TOF/MS by using 9-aminoacridine as a matrix. The standard calibration curves showed that choline and PA could be detected with linearity over the range from 0.05 and 1 pmol, respectively. Importantly, this method enables the concomitant detection of choline and PA as a reaction product of PC hydrolysis by PLD2 proteins. Thus, our simple and direct method would be useful to characterize the enzymatic properties of PLD, thereby providing insight into mechanisms of PLD activation.