Retention time scheduling for MRM transitions during targeted assays enables more compounds to be quantified with higher quality results. In this assay, MRMs to quantitate over 1150 lipid molecular species were combined into a single assay, covering lipids containing fatty acids with 14 to 22 carbons and 0 to 6 double bonds. The selected amide column chemistry provides reproducible isomer separation based on lipid class, to minimize isomeric overlap. This liquid chromatography separation strategy is then coupled with the time scheduled targeted MRM assay. To validate this method, we selected DES1 knockout mice to measure lipid changes in liver and adipose tissues. The method was then adjusted to optimize window width and dwell weight to enhance sensitivity and coverage. This method provides extensive, reproducible lipid coverage in complex biological samples like tissue, cells, and plasma.
Liver, epididymal white adipose tissue (eWAT), and subcutaneous white adipose tissue (sWAT) extracts were subjected to amide LC-MS/MS analysis with a QTRAP® 6500+ mass spectrometer coupled with ExionLC™ System. To streamline the development of this assay, an Excel based method optimization tool (sMRM Pro Builder) has been developed to assist with the assay refinement steps. The method is customizable, so new lipid categories, classes, and molecular species can be added to the MRMs list.
Liver, eWAT, and sWAT tissues were harvested from dihydroceramide desaturase-1 (DES1) knockout mice. DES1 is the enzyme responsible for inserting the 4,5-trans-double bond into the sphingolipid backbone causing dihyrdoceramides conversion to ceramides.
This widely targeted panel allows for quantitation of 19 lipid classes in several matrices, and yet has the sensitivity and selectivity to visualize significant changes in lipid species that are low abundant in eWAT and liver tissue.