Reversed-Phase LC/MS Method for Polyphosphoinositide Analyses: Changes in Molecular Species Levels during Epidermal Growth Factor Activation in A431 Cells
Reversed-Phase LC/MS Method for Polyphosphoinositide Analyses: Changes in Molecular Species Levels during Epidermal Growth Factor Activation in A431 Cells
复制标题
DOI：
10.1021/ac801451p
复制
发表时间：
2008-12-01
影响因子：
7.4
通讯作者：
Taguchi, Ryo
中科院分区：
文献类型：
--
作者：
Ogiso, Hideo;Taguchi, Ryo
在脂质代谢组学的研究中，液相色谱/质谱（LC/MS）是一种强大而流行的技术。虽然已经建立了有效的反相（RP） LC/MS分离磷脂分子种类的方法，但用于分析磷脂酰肌醇磷酸（PIPs）的RPLC方法尚未见报道。在本研究中，我们开发了适合PIP分析的条件。结合（二乙胺）乙基(DEAE)-纤维素预处理，每个类似于6 × 10(6)个培养细胞的磷脂酰肌醇单磷酸（PIP I），二磷酸（PIP2）和三磷酸标准物至少可测量1 pmol。利用这些方法，我们在表皮生长因子（EGF）刺激的A431细胞（一种人表皮样癌细胞系）中检测到超过12种PIP1的浓度升高。刺激后检测到的PIP2种类没有升高。我们还使用另一组方法检测到egf诱导的几种磷脂酸水平的增加。我们的方法灵敏地测定了生物样品中的pip，因此适用于磷酰苷代谢的分析。
In studies on lipid metabolomics, liquid chromatography/mass spectrometry (LC/MS) is a robust and popular technique. Although effective reversed-phase (RP) LC/MS methods enabling the separation of phospholipid molecular species have been developed, RPLC methods to analyze phosphatidylinositol phosphates (PIPs) have not been reported. In this study, we developed conditions suitable for PIP analysis. Coupled with (diethylamino)etbyl (DEAE)-cellulose pretreatment, at least 1 pmol each of phosphatidylinositol monophosphates (PIP I), bisphosphates (PIP2), and triphosphates standards per similar to 6 x 10(6) cultured cells could be measured. Using these methods, we detected elevated concentrations of more than 12 PIP1 species in epidermal growth factor (EGF)stimulated A431 cells, a human epidermoid carcinoma cell line. The PIP2 species detected were not elevated after stimulation. We also detected EGF-induced increases in the levels of several phosphatidic acid species using another set of methods. Our method sensitively determined PIPs within a biological sample and is thus suitable for analysis of phoisphoiniositide metabolism.