Directed Non-targeted Mass Spectrometry and Chemical Networking for Discovery of Eicosanoids and Related Oxylipins

Abstract

Eicosanoids and related species are critical, small bioactive mediators of human physiology and inflammation. While ~1100 distinct eicosanoids have been predicted to exist, to date, less than 150 of these molecules have been measured in humans, limiting our understanding of eicosanoids and their role in human biology. Using a directed non-targeted mass spectrometry approach in conjunction with computational chemical networking of spectral fragmentation patterns, we find over 500 discrete chemical signals highly consistent with known and putative eicosanoids in human plasma, including 46 putative novel molecules not previously described, thereby greatly expanding the breath of prior analytical strategies. In plasma samples from 1500 individuals, we find members of this expanded eicosanoid library hold close association with markers of inflammation, as well as clinical characteristics linked with inflammation, including advancing age and obesity. These experimental and computational approaches enable discovery of new chemical entities and will shed important insight into the role of bioactive molecules in human disease.

Plasma Docosahexaenoic Acid and Eicosapentaenoic Acid Concentrations Are Positively Associated with Brown Adipose Tissue Activity in Humans

“…Lipidomic analyses were conducted on plasma samples between 1 and 2 years after sample collection. While stability of the analytes was not evaluated in this study, previous publications have determined that long-term storage at −80 °C has minimal effect on oxylipin and NEFA concentrations [ 27 , 28 , 29 ], even in the absence of antioxidants. Reagents were purchased from Sigma-Aldrich (Sigma-Aldrich, Castle-Hill, NSW, Australia) unless specified.…”

Methods of the Analysis of Oxylipins in Biological Samples

“…Recently, Watrous et al developed a new approach for using non-targeted LC-MS/MS to classify putative known oxylipins. Based on the fragmentation patterns obtained from a large number of standards, they maintained a network due to which it was possible to classify more than 500 different oxylipins in human plasma [169]. It is also possible to combine both approaches like Wheelock et al, who used targeted (QqQ) metabolomics to identify 36 oxylipins and non-targeted (orbitrap) to annotate 219 metabolites [170].…”

Metabolomic profiles associated with a mouse model of antipsychotic-induced food intake and weight gain

“…Metabolomics analysis was performed on collected mouse plasma and hypothalamus samples as previously described 31 – 33 . Briefly, small bioactive lipid metabolites and polar, hydrophilic metabolites were extracted from plasma samples using addition of organic solvent followed by offline solid phase extraction (SPE), as described 31 – 33 .…”

“…Metabolomics analysis was performed on collected mouse plasma and hypothalamus samples as previously described 31 – 33 . Briefly, small bioactive lipid metabolites and polar, hydrophilic metabolites were extracted from plasma samples using addition of organic solvent followed by offline solid phase extraction (SPE), as described 31 – 33 . Metabolites were chromatographically separated using a Thermo Vanquish UPLC system with a Phenomenex Kinetex C18 (1.7um particle size, 100 × 2.1 mm) or Merck-SeQuant ZIC-pHILIC (5 μm particle size, 100 × 2.1 mm) column for measure of bioactive lipids and polar metabolites, respectively.…”

“…Metabolites were chromatographically separated using a Thermo Vanquish UPLC system with a Phenomenex Kinetex C18 (1.7um particle size, 100 × 2.1 mm) or Merck-SeQuant ZIC-pHILIC (5 μm particle size, 100 × 2.1 mm) column for measure of bioactive lipids and polar metabolites, respectively. Mass spectra were acquired on a Thermo QExactive orbitrap mass spectrometer with electrospray ionization in negative mode and positive mode, for measure of bioactive lipids and polar metabolites, respectively, as previously described 31 – 33 . Metabolites were identified by matching accurate mass, retention time, and MS/MS fragmentation patterns to an in-house library of commercially available standards, as described 31 – 33 .…”