The study of complex lipids is critical to gaining insight across a variety of biological processes. From the structure of biological membranes to signaling lipids, to energy storage, the identification and quantification of complex lipids reveal key understandings of the physiology and mechanisms underlying metabolic diseases.
The Basics of Complex Lipid Structure
Complex lipids are extraordinarily diverse with thousands of unique species. Complex lipids have a modular structure, consisting of lipid classes consisting of a head group, which defines the class, and one or more fatty acid moieties. Together, a matrix is constructed with lipid classes forming the columns and fatty acids forming the rows. Concentration can then be calculated by the sum of each row for fatty acids and the sum of each column for lipid classes.
Challenges and Essentials for Lipidomics
Given the diversity in lipid structure, lipidomics faces several challenges. Due to a large number of isomers, exact mass is not sufficient to identify lipids. Moreover, many isomers are not readily separated by chromatography and many standards are not commercially available. The matrix structure of complex lipids presents three essentials for accurate lipidomic profiling that come with their own challenges:
- Specificity—The lipidomic profiling method must accurately identify the fatty acid constituent of the lipid. Many methods fail to resolve fatty acid composition, and non-specific methods do not allow mapping of lipid species to the matrix.
- Quantitation—The lipidomic profiling method must provide accurate summing of rows and columns. In other words, the molar concentration of each lipid species is required. Quantitation using relative methods does not provide accurate measures for lipid class or fatty acids.
- Coverage—The lipidomic profiling method must provide broad coverage. A partially completed matrix will not provide an accurate representation of the contribution of a lipid class or fatty acid.
Metabolon’s Complex Lipids Targeted Panel
Metabolon’s solution for these challenges is the Complex Lipids Targeted Panel. Developed in partnership with scientists from SCIEX, the panel consists of several analytical steps designed for confident and accurate identification and quantitation of hundreds of lipids.
The Complex Lipids Targeted Panel begins by adding proprietary internal standard mixure designed to allow for differing extraction and ionization efficiencies to the sample before lipid extraction. The panel is capable of delivering quantitative values. Next, the lipid-containing solution is infused into the ionization source of a QTRAP 5500 mass spectrometer. After ionization, the lipids are passed through SelexION differential mobility separation (DMS). Since SelexION separates lipids by class through the selective passage of specific lipid classes, this minimizes isobaric overlaps. Following DMS, lipids enter multiple reaction monitoring (MRM) where the impact mass of the lipid and the mass of the characteristic fragments are measured, allowing for the identification of fatty acid side chains. Finally, quantitation occurs using more than 50 isotopically labeled internal standards, permitting accurate quantitation across and within lipid classes. Intensity data are collected and calculated to obtain a molar concentration of each lipid.
The Complex Lipids Targeted Panel not only meets key lipidomic challenges, but is also high throughput, providing broad coverage of over 1,100 typical mammalian lipids. At least 800 of these species are detected in human plasma, achieved without sacrificing speed (total runtime: 16 min/sample).
Insights Gained from Metabolon’s Complex Lipids Targeted Panel
An excellent demonstration of how Metabolon’s Complex Lipids Targeted Panel fulfills the essentials of lipidomic profiling can be seen in a report from Wu and colleagues.1 In this study, published in Nature Communications, the researchers divided acutely injured patients into two groups. One group of 84 patients was provided with two units of thawed allogeneic plasma (TP) while the control group of 109 patients was given standard of care alone. A separate group of 17 healthy volunteers also had their plasma collected as baseline controls. From these patients, the Prehospital Air Medical Plasma (PAMPer) biobank was generated. Blood samples were collected upon admission, 24 hours after admission, and 72 hours after admission. As the study progressed, the patients were then stratified into three groups: those that didn’t survive, others who survived but failed to resolve, and those who recovered.
After collecting plasma from the patient population, the researchers used the Metabolon Complex Lipids Targeted Panel to generate lipid-based profiles as patients underwent their respective treatment procedures. Initial liquid chromatography-mass spectrometry (LC-MS) efforts with this panel yielded 996 quantifiable lipids across all patients. The researchers then found that the total abundance of these lipids was significantly lower among the non-survivors within the first day of treatment than the healthy controls and non-resolving patients. TP treatment increased patient survival and also restored lipid levels to those similar to healthy controls.
The concentrations of a subset of lipids also increased among non-resolving survivors within 72 hours. Most notably, the authors observed an increased abundance of phosphatidylethanolamines (PE) and ceramides (CER) after 72 hours of treatment among these patients. Based on the trends observed from the lipidomics study results, the researchers generated a lipid reprogramming score (LRS) to predict instances of worse outcomes for acute critical illness patients. LRS was successful, being strongly prognostic for worsened clinical outcomes among acute trauma and COVID-19 patients.
This report is an excellent demonstration of the capabilities of the Complex Lipids Targeted Panel—in addition to individual lipid species, quantitative data on lipid class and fatty acid composition and concentration provide valuable insight and robust data sets.
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References
- Wu J, Cyr A, Gruen DS, et al. Lipidomic signatures align with inflammatory patterns and outcomes in critical illness. Nat Commun. 2022;13(1):6789. Published 2022 Nov 10. doi:10.1038/s41467-022-34420-4
