Qualitative Characterization of the Rat Liver Mitochondrial Lipidome using LC-MS Profiling and High Energy Collisional Dissociation (HCD) All Ion Fragmentation.

Lipids play multiple roles essential for proper mitochondrial function, from their involvement in membrane structure and fluidity, cellular energy storage, and signaling. Lipids are also major targets for reactive species, and their peroxidation byproducts themselves mediate further damage. Thousands of lipid species, from multiple classes and categories, are involved in these processes, suggesting lipid quantitative and structural analysis can help provide a better understanding of mitochondrial physiological status. Due to the diversity of lipids that contribute to and reflect mitochondrial function, analytical methods should ideally cover a wide range of lipid classes, and yield both quantitative and structural information. We developed a high resolution LC-MS method that is able to monitor the major lipid classes found in biospecimens (ie. biofluids, cells and tissues) with relative quantitation in an efficient, sensitive, and robust manner while also characterizing individual lipid side-chains, by all ion HCD fragmentation and chromatographic alignment. This method was used to profile the liver mitochondrial lipids from 192 rats undergoing a dietary macronutrient study in which changes in mitochondria function are related to changes in the major fat and glycemic index component of each diet. A total of 381 unique lipids, spanning 5 of the major LIPID MAPS defined categories, including fatty acyls, glycerophospholipids, glycerolipids, sphingolipids and prenols, were identified in mitochondria using the non-targeted LC-MS analysis in both positive and negative mode. The intention of this report is to show the breadth of this non-targeted LC-MS profiling method with regards to its ability to profile, identify and characterize the mitochondrial lipidome and the details of this will be discussed.