High‐throughput Targeted Lipidomics Analysis of Dihydroceramide Desaturase‐1 (DES1) Knockout Mice

Abstract

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. The selected amide column chemistry provides reproducible isomer separation based on lipid class. This Liquid Chromatography separation strategy, is then coupled with the time scheduled targeted assay. To validate this method, we selected DES1 knockout mice to measure lipid changes in liver and adipose tissues. The MRM™ Pro Builder tool (SCIEX) was then used to optimize window width and dwell weight to enhance sensitivity and coverage of method. This method provides extensive, reproducible lipid coverage in complex biological samples like tissue, cells, and plasma.Amide column chemistry was chosen for lipid class separation and minimize isomeric interference. The target list of lipids is comprehensive, covering most major lipid classes and categories, and MRMs were selected to cover lipids containing fatty acids with 14 to 22 carbons and 0 to 6 double bonds. The method is customizable, so new lipid categories, classes and molecular species can be added to the MRMs list. This method provides quantitative measurement of over 1150 different lipid molecular species in a rapid, highly reproducible manner. The sMRM Pro Builder template which was developed to streamline the method optimization process, enables assigning the retention time, optimize dwell weight and set window size per MRM to enhanced coverage and sensitivity of the method. This optimization improved results quality especially on low abundant lipids.Liver and eWAT 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 the dihydroceramide conversion to ceramide. While both of these classes of lipids are lower in abundance in the chosen tissues, this method shows significant changes in these lipid classes in a quantitative manner. However, lipids from another 17 classes were not changing.Lipid standards from 19 different classes, which are either heavy isotopic labeled lipids or odd chain lipids, served as internal standard. This method provided extensive lipid class coverages including, CE, CER, DCER, HCER, LCER, TAG, DAG, MAG, LPC, PC, LPE, PE, LPG, PG, LPI, PI, LPS and PS.