Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan.
Anal Chem. 2010 Dec 1;82(23):9858-64. doi: 10.1021/ac102211r. Epub 2010 Nov 9.
Normal-phase or reverse-phase liquid chromatography has been used in phospholipidomics for lipid separation prior to mass spectrometry analysis. However, separation using a single separation mode is often inadequate, as high-abundance phospholipids can mask large numbers of low-abundance lipids of interest. In order to detect and quantify low-abundance phospholipids, we present a novel two-dimensional (2D) approach for sensitive and quantitative global analysis of phospholipids. The methodology monitors individual glycerolipids and phospholipids through the use of a new quantitative normal-phase, solid-phase extraction procedure, followed by molecular characterization and relative quantification using an ion-trap Orbitrap equipped with a reverse-phase liquid chromatograph, with data processing by MS++ software. The CV (%) of the peak area of each lipid standard was less than 15% with this extraction method. When the method was applied to a liver sample, we could detect more phosphatidylserine (PS) compared to the previous method. Finally, our developed method was applied to Alzheimer's disease (AD) plasma samples. Several hundred peaks were detected from a 60 μL plasma sample. A partial-least-squares discriminant analysis (PLS-DA) plot using peak area ratio gave a unique group of PLS scores which could distinguish plasma samples of Alzheimer's disease (AD) patients from those of age-matched healthy controls.
正相或反相液相色谱法已用于磷脂组学中，在进行质谱分析之前对脂质进行分离。然而，单一分离模式的分离往往不够充分，因为高丰度的磷脂会掩盖大量感兴趣的低丰度脂质。为了检测和定量低丰度磷脂，我们提出了一种新的二维（2D）方法，用于灵敏和定量的全局磷脂分析。该方法通过使用新的定量正相、固相萃取程序来监测个体甘油磷脂和磷脂，然后使用配备反相液相色谱的离子阱轨道阱进行分子特征分析和相对定量，并通过 MS++软件进行数据处理。使用这种提取方法，每个脂质标准品的峰面积的 CV（%）小于 15%。当将该方法应用于肝样品时，与以前的方法相比，我们可以检测到更多的磷脂酰丝氨酸（PS）。最后，我们开发的方法应用于阿尔茨海默病（AD）血浆样品。从 60μL 血浆样品中检测到数百个峰。使用峰面积比的偏最小二乘判别分析（PLS-DA）图给出了一个独特的 PLS 得分组，可将阿尔茨海默病（AD）患者的血浆样本与年龄匹配的健康对照者的血浆样本区分开来。
