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. 2017 Nov;58(11):2229-2237.
doi: 10.1194/jlr.D077123. Epub 2017 Sep 5.

Comprehensive analyses of oxidized phospholipids using a measured MS/MS spectra library

Ryohei Aoyagi  1   2 , Kazutaka Ikeda  1   2 , Yosuke Isobe  1   2 , Makoto Arita  3   2   4
Affiliations

Comprehensive analyses of oxidized phospholipids using a measured MS/MS spectra library

Ryohei Aoyagi et al. J Lipid Res. 2017 Nov.

Abstract

Oxidized phospholipids (OxPLs) are widely held to be associated with various diseases, such as arteriosclerosis, diabetes, and cancer. To characterize the structure-specific behavior of OxPLs and their physiological relevance, we developed a comprehensive analytical method by establishing a measured MS/MS spectra library of OxPLs. Biogenic OxPLs were prepared by the addition of specific oxidized fatty acids to cultured cells, where they were incorporated into cellular phospholipids, and untargeted lipidomics by LC-quadrupole/TOF-MS was applied to collect MS/MS spectra for the OxPLs. Based on the measured MS/MS spectra for about 400 molecular species of the biogenic OxPLs, we developed a broad-targeted lipidomics system using triple quadrupole MS. Separation precision of structural isomers was optimized by multiple reaction monitoring analysis and this system enabled us to detect OxPLs at levels as low as 10 fmol. When applied to biological samples, i.e., mouse peritoneal macrophages, this system enabled us to monitor a series of OxPLs endogenously produced in a 12/15-lipoxygenase-dependent manner. This advanced analytical method will be useful to elucidate the structure-specific behavior of OxPLs and their physiological relevance in vivo.

Keywords: broad-targeted lipidomics; lipidomics; lipoxygenase; mass spectrometry; oxidized lipids; tandem mass spectrometry; untargeted lipidomics.

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Figures

Fig. 1.
Fig. 1.
2D map of mass versus LC retention time of lipids isolated from control and 12-HETE-treated cells. The 2D map has the m/z values of precursor ions along the vertical axis and the retention times (RT) along the horizontal axis. A: Plots of precursor ions identified as lyso-PLs, PLs, and sphingolipids. B: Plots of candidate signals for PLs containing 12-HETE determined by the presence of a fragment ion (m/z 319.2) corresponding to AA+O.
Fig. 2.
Fig. 2.
Structural identification of OxPLs by MS/MS fragmentation patterns. Structural analysis of candidate signals obtained from a series of biogenic OxPLs. Based on the MS/MS spectra, the OxPL structures were assigned to the corresponding fragments of the acyl chains, the diagnostic ions of molecular species of oxidized fatty acids, and the head groups or neutral loss ions of the head groups. GPhE, phosphoglycerol ethanolamine; LPE, lyso-PE; PhI, phosphoinositol; NL, neutral loss.
Fig. 3.
Fig. 3.
OxPL detection by LC tripleQ MS with optimized MRM transitions and LC gradient conditions. A: A series of biogenic OxPCs containing HETEs were selectively detected by optimized MRM transitions. B: Structural isomers were separated by the optimized LC gradient condition.
Fig. 4.
Fig. 4.
Calibration curves for quantification of OxPLs. OxPL standard solutions corresponding to 10, 20, 50, 100, 200, and 500 fmol were quantified by LC tripleQ MS with optimized MRM transitions. Calibration curves were constructed by plotting the peak areas and concentrations.
Fig. 5.
Fig. 5.
Quantification of OxPLs in mouse peritoneal macrophages. Peritoneal macrophages isolated from WT and 12/15-LOX KO mice were analyzed. Levels of OxPLs were expressed as concentration per 1.0 × 106 cells. (n = 3, mean ± SE, *P < 0.05, **P < 0.01).
See this image and copyright information in PMC

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