Chapter 24 Qualitative Characterization of the Rat Liver Mitochondrial Lipidome Using All Ion Fragmentation on an Exactive Benchtop Orbitrap MS Irina G. Stavrovskaya, Rose M. Gathungu, Susan S. Bird, and Bruce S. Kristal Abstract Untargeted lipidomics profiling by liquid chromatography–mass spectrometry (LC-MS) allows researchers to observe the occurrences of lipids in a biological sample without showing intentional bias to any specific class of lipids and allows retrospective reanalysis of data collected. Typically, and in the specific method described, a general extraction method followed by LC separation is used to achieve nonspecific class coverage of the lipidome prior to high resolution accurate mass (HRAM) MS detection. Here we describe a workflow including the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition. We also highlight how, in this method, all ion fragmentation can be used to identify species of lower abundances, often missed by data dependent fragmentation techniques. Here we describe the isolation of mitochondria from liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for data acquisition. Key words Mitochondria, Lipidomics, LC-MS, Cardiolipins, Lysophospholipids, HCD 1 Introduction Mitochondria are intracellular membrane-enclosed organelles that play crucial role in bioenergetics, biosynthesis of critical cellular constituents, the regulation of cell survival, and the execution of cell death pathways [1, 2]. Lipids are essential to mitochondrial viability, and they are involved in the regulation of the wide range of mitochondrial functions, such as maintenance of membrane struc- tural composition and fluidity, membrane fusion and fission, elec- tron transport and oxidative phosphorylation, signal transduction, interaction with other cellular constituencies such as proteins and glycoproteins, and energy storage [3–5]. The role of lipids in mitochondrial function is, for example, exemplified by mitochon- drial phospholipid cardiolipin (CL). CL, which is almost exclusively Volkmar Weissig and Marvin Edeas (eds.), Mitochondrial Medicine: Volume 1: Targeting Mitochondria, Methods in Molecular Biology, vol. 2275, https://doi.org/10.1007/978-1-0716-1262-0_24, © Springer Science+Business Media, LLC, part of Springer Nature 2021 379