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Characterization of lysophosphatidic acid subspecies produced by autotaxin using a modified HPLC ESI-MS/MS method
- Wijesinghe, Dayanjan S., Mayton, Eric K., Mietla, Jennifer A., Mukherjee, Abir, Wu, Jinhua, Fang, Xianjun, Chalfant, Charles E.
- Analytical methods 2011 v.3 no.12 pp. 2822-2828
- bioassays, blood serum, cell culture, cell lines, cell viability, electrospray ionization mass spectrometry, high performance liquid chromatography, humans, lipids
- 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.