Comprehensive Lipidomics Analysis of Bioactive Lipids in Complex Regulatory Networks

摘要

In the present work we describe the development of annanalytical technique for simultaneous profiling of over 100nbiochemically related lipid mediators in biological samples.nA multistep procedure was implemented to extractneicosanoids and other bioactive lipids from the biologicalnmatrix, chromatographically separate them using fastnreversed-phase liquid chromatography, tentatively identifynnew candidate eicosanoids through a matching processnof retention times, isotope distribution patterns, and highresolutionnorbitrap MS/MS fragmentation patterns, andnsubsequently quantify tentative candidates by means ofnanalytical reference standards. Key new aspects of thisnprofiling technique included the classification of bioactivenlipids into 12 groups according to their calculated exactnmasses and the development of optimized liquid chromatographicnconditions for these groups to achieve sufficientnseparation of the numerous isobaric and isomericnspecies, many of which exhibited virtually identical collision-ninduced dissociation behavior. Importantly, nonanalytical standards were required at this screening stagenof the assay, and tentative identifications were achievednby matching results to selected reference species fromneach of the groups. The analytical figures of merit for thenorbitrap assay such as linear dynamic range, limit ofndetection, limit of quantitation, and precision demonstratednthat the performance of the assay was very similarnto that of a quadrupole linear ion trap assay, which wasnused for validation purposes. The method allowed us tonexamine eicosanoid profiles within the signaling cascadenin chronic lymphocytic leukemia (CLL) cells under basalnconditions and following arachidonic acid stimulation. Thenpreliminary screening based on high-resolution tandemnmass spectrometry data along with isotope pattern andnretention time matching revealed the presence of 15nbioactive lipids, belonging to a range of prostaglandin,nleukotriene, and hydroxy and epoxy fatty acid lipid mediatorsnproduced by CLL cells.