Characterization of lysophosphatidic acid subspecies produced by autotaxin using a modified HPLC ESI-MS/MS method.
Characterization of lysophosphatidic acid subspecies produced by autotaxin using a modified HPLC ESI-MS/MS method.
复制标题
DOI：
10.1039/c1ay05459g
复制
发表时间：
2011-11-01
期刊：
影响因子：
--
通讯作者：
Chalfant CE
中科院分区：
文献类型：
--
作者：
Wijesinghe DS;Mayton EK;Mietla JA;Mukherjee A;Wu J;Fang X;Chalfant CE
溶血磷脂酸 (LPA) 是一种生物活性脂质，具有多种生物学功能，包括在细胞存活、增殖和迁移中的作用。虽然高效液相色谱电喷雾电离串联质谱 (HPLC ESI-MS/MS) 技术已用于测量人体血液、血清和血浆中的 LPA 水平，但目前的方法无法轻松检测细胞培养物中的微量 LPA 水平。在本研究中，开发了一种改进的 HPLC ESI-MS/MS 方法，其灵敏度更高，可以精确测量 LPA 水平，定量限约为 10 飞摩尔。通过对先前表征的异位表达自分泌运动因子的细胞系的培养基中 LPA 水平进行定量来验证该方法。具体来说，自分泌运动因子过度表达会诱导 16:0、18:2、18:1、18:0 和 20:4 LPA 亚种的增加，但不会诱导 22:6 LPA 亚种的增加。最后，该 HPLC ESI-MS/MS 方法通过之前用于测定 LPA 水平的生物测定进行了交叉验证。因此，这种 HPLC ESI-MS/MS 方法将允许研究人员测量体外 LPA 水平，并区分特定的 LPA 亚种以描述个体生物学机制。
Lysophosphatidic acid (LPA) is a bioactive lipid with a plethora of biological functions including roles in cell survival, proliferation, and migration. Although high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC ESI-MS/MS) technology has been used to measure the levels of LPA in human blood, serum and plasma, current methods cannot readily detect the minute levels of LPA from cell culture. In this study, a modified HPLC ESI-MS/MS method with enhanced sensitivity was developed, which allows accurate measurements of LPA levels with a limit of quantitation at approximately 10 femtomoles. The method was validated by quantitation of LPA levels in the media of previously characterized cell lines ectopically expressing autotaxin. Specifically, autotaxin overexpression induced an increase in the 16:0, 18:2, 18:1, 18:0, and 20:4 subspecies of LPA, but not the 22:6 LPA subspecies. Lastly, this HPLC ESI-MS/MS method was cross-validated via biological assays previously utilized to assay LPA levels. Hence, this HPLC ESI-MS/MS method will allow researchers to measure in vitro LPA levels and also distinguish between specific LPA subspecies for the delineation of individual biological mechanisms.