Targeted lipidomics on the QTRAP® 6500+ System
Mackenzie Pearson1, Santosh Kapil Kumar Gorti1, Trevor S. Tippets2; Bhagirath Chaurasia2; Scott A. Summers2
1SCIEX, USA; 2University of Utah, Dept of Nutrition and Integrative Physiology, Salt Lake City, UT
Previously a targeted lipid profiling method was developed for quantifying a many lipid molecular species (~1500) in a single run. To determine the performance of the method in biological matrices, a short feasibility study was done here using LC-MSM on the QTRAP 6500+ system. Using well characterized DES1 knockout mouse model, the expected changes in ceramides and dihydroceramides were observed. Many lipid classes were not found to be changing (17 classes) relative to the control.
Targeted lipidomics approach using liquid chromatography (LC) coupled with triple quadrupole-based instrumentation in the multiple reaction monitoring (MRM) mode provides high sensitivity and specificity. Amide column separation is an attractive chromatographic strategy that effectively resolves both polar and non-polar lipids into classes and subclasses, which is critical for reliable lipid identification and quantification.
A targeted global lipid profiling strategy has been developed, enabling a broad array of different lipids to be quantified at the molecular species level (~1150 molecular species). 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 14-22 carbons and 0-6 double bonds. The optimized chromatography method was developed to minimize isobaric interference through the chromatographic separation of lipid class. The method is customizable, so new lipid categories, classes, and molecular species can be added.
To validate this method, which allows for a relatively rapid and specific lipid screening technique, a DES1 knockout mice model was selected. A small feasibility study was performed to measure lipid changes in liver and adipose tissues to determine if expected changes could be measured.
Sample preparation: Liver and epidydimal white adipose tissue (eWAT) was homogenized in cold methanol and extracted using the Bligh-Dyer method2, n=8 and 7 for wildtype and knockout, respectively. The extracts were diluted 100-fold in ethanol before injection. Avanti SPLASH standard mixture was spiked at 1:10 ratio. The volume of Lipidyzer™ Platform standards were calculated using the Lipidomics Workflow Manager. Accurate quantitation (less than 10% quantitative bias) for most classes of lipids analyzed can be achieved by using the Lipidyzer Platform standards. Classes not included were relatively quantified with the addition of SPLASH standards.
LC-MS analysis: LC separation was performed using an ExionLC™ System consisting of a binary high pressure mixing gradient pump with degasser, a thermostated autosampler, and a column oven (35 ºC). The QTRAP 6500+ System, equipped with an IonDrive™ Turbo V source, was operated in low mass mode with polarity switching. Retention times are first roughly determined using either the BHE with spiked internal standards or a representative matrix sample and the unscheduled MRM method. Analysis of this preliminary data enables the development of a time-scheduled MRM method leveraging the enhanced features in the Scheduled MRM Algorithm Pro. Details on the chromatographic and MS methods can be obtained from the Comprehensive Targeted Method for Global Lipidomics Screening. 1
Data processing: All data was processed using MultiQuant™ Software 3.0.2. Automated computation of the time scheduled final MRM methods was performed using the sMRM Pro Builder 1.1. 1
Lipid profiling using the targeted LC-MRM method was performed on both liver and epidydimal white adipose tissue obtained from the control and knockout mice. Many lipids were measured across a broad range of lipid classes (Figure 2). Many were found to have minimal changes in abundance (Figure 3). Of interest in this model system were the changes to the ceramides and dihydroceramides (Figure 4).2 They are expressed in very low levels in adipose tissue but were easily measured with this sensitive, targeted approach.
Here a short feasibility study was performed to test the global lipid profiling methodology using LC-MRM on a QTRAP 6500+ System using two tissue types in mouse models. A DES1 knockout model was chosen to validate the method as changes were expected in the CER and DCER species.