Haag Mathias, Schmidt Angelika, Sachsenheimer Timo, Brügger Britta
Heidelberg University Biochemistry Center, University of Heidelberg, Heidelberg, Germany.
Tumorimmunology Program, Division of Immunogenetics (D030), German Cancer Research Center (DKFZ), Heidelberg, Germany.
Metabolites. 2012 Jan 16;2(1):57-76. doi: 10.3390/metabo2010057.
Lipids, such as phosphoinositides (PIPs) and diacylglycerol (DAG), are important signaling intermediates involved in cellular processes such as T cell receptor (TCR)-mediated signal transduction. Here we report identification and quantification of PIP, PIP2 and DAG from crude lipid extracts. Capitalizing on the different extraction properties of PIPs and DAGs allowed us to efficiently recover both lipid classes from one sample. Rapid analysis of endogenous signaling molecules was performed by nano-electrospray ionization tandem mass spectrometry (nano-ESI MS/MS), employing lipid class-specific neutral loss and multiple precursor ion scanning for their identification and quantification. Profiling of DAG, PIP and PIP2 molecular species in primary human T cells before and after TCR stimulation resulted in a two-fold increase in DAG levels with a shift towards 1-stearoyl-2-arachidonoyl-DAG in stimulated cells. PIP2 levels were slightly reduced, while PIP levels remained unchanged.
脂质，如磷酸肌醇（PIPs）和二酰基甘油（DAG），是参与细胞过程（如T细胞受体（TCR）介导的信号转导）的重要信号中间体。在此，我们报告了从粗脂质提取物中鉴定和定量PIP、PIP2和DAG的方法。利用PIPs和DAGs不同的提取特性，我们能够从一个样品中高效回收这两类脂质。通过纳米电喷雾电离串联质谱（nano-ESI MS/MS）对内源性信号分子进行快速分析，采用脂质类别特异性中性丢失和多前体离子扫描进行鉴定和定量。对原发性人类T细胞在TCR刺激前后的DAG、PIP和PIP2分子种类进行分析，结果显示刺激细胞中DAG水平增加了两倍，并向1-硬脂酰-2-花生四烯酰-DAG转变。PIP2水平略有降低，而PIP水平保持不变。
