Shotgun lipidomics of cardiolipin molecular species in lipid extracts of biological samples

Abstract

Cardiolipin is a prominent component of the mitochondrial inner membranes contributing to the regulation of multiple discrete mitochondrial functions. Here, we extend shotgun lipidomics to identify and quantitate cardiolipin molecular species directly from lipid extracts of biological samples. Three shotgun lipidomics approaches for analyses of cardiolipin molecular species were developed using either a continuous ion-transmission instrument (i.e., triple-quadrupole type) with either low or high mass resolution settings or a high mass resolution hybrid pulsed instrument [i.e., quadrupole time-of-flight (QqTOF) type]. Three chemical principles were used for the development of these approaches. These include the marked enrichment of linoleate in cardiolipin to maximize the signal-to-noise ratio, the specific neutral loss of ketenes from doubly charged cardiolipin molecular ions to yield doubly charged triacyl monolysocardiolipins, and the doubly charged character of two phosphates in each cardiolipin molecular species. Through these techniques, we identified and quantified the specific molecular species profiles of cardiolipin directly from lipid extracts of mouse heart, liver, and skeletal muscle. The accuracy (z5%) and the low end of the linear dynamic range (10 fmol/ml) for quantitation make these approaches useful for studying alterations in cardiolipin metabolism in multiple disease states using either type of mass spectrometer.-Han, X., K. Yang, J. Yang, H. Cheng, and R. W. Gross. Shotgun lipidomics of cardiolipin molecular species in lipid extracts of biological samples. J. Lipid Res. 2006. 47: 864-879. Supplementary key words electrospray ionization mass spectrometry . mitochondria . multidimensional mass spectrometry Cardiolipin (1,3-diphosphatidyl-sn-glycerol) is a unique class of anionic phospholipids, because each molecular species is composed of a dimer of two phosphatidyl moieties, three chiral centers in three distinct glycerol moieties, and four fatty acyl chains. Cardiolipin is predominantly, if not nearly exclusively, present in mitochondrial membranes in eukaryotic cells (1, 2), and its genetic ancestry can be traced back to bacterial membranes. Thousands of distinct molecular species, including regioisomers, would be present in the cellular membranes if all naturally occurring fatty acids were randomly incorporated into cardiolipin molecular species (3). However, biological organisms selectively use a very limited array of fatty acids for the biosynthesis of cardiolipin molecular species in a stereoselective manner that is species-, organ-, and cell type-specific. This is accomplished through remodeling enzymes (e.g., the tazaffins), which exist in multiple splice variants. In heart, linoleate is present predominantly in the acyl chains of cardiolipin molecular species (3, 4). Therefore, in heart, cardiolipin represents an enriched storage depot of linoleic acid relative to other phospholipid classes. The biochemical mechanisms underlying the production of linoleate-enriched cardio...

Genetic Ablation of Calcium-independent Phospholipase A2γ Leads to Alterations in Hippocampal Cardiolipin Content and Molecular Species Distribution, Mitochondrial Degeneration, Autophagy, and Cognitive Dysfunction

“…Peak heights represent the relative intensity (%) of the individual molecular species to the intensity of the CL internal standard. The asterisks indicate doubly charged CL plusone isotopologues whose ion peak intensities were utilized to quantify individual CL molecular species as described previously (37,78). The results indicate an increase in multiple CL species in the iPLA 2 ␥ Ϫ/Ϫ mice and especially those containing shorter chain length aliphatic chains.…”

“…Enhanced shotgun lipidomics analyses of CL were performed using the Mϩ1/2 isotopologue approach with a mass resolution setting of 0.3 Th as previously described (37). Typically, a 2-to 5-min period of signal averaging was employed for each mass spectrum.…”

Dynamic simulation of cardiolipin remodeling: greasing the wheels for an interpretative approach to lipidomics

“…E: A representative mouse myocardial cardiolipin distribution as determined by MDMS-SL. The asterisks indicate the individual ion peaks of mouse myocardial cardiolipin (15). The mouse myocardial cardiolipin molecular species detected and quantifi ed by MDMS-SL as well as determined by the dynamic simulation were tabulated in Table 1 .…”

“…The pellet was resuspended in HM buffer without BSA, and the protein concentration of the resuspended mitochondrial fractions was determined using the BCA protein assay (Thermo Scientifi c, Rockford, IL). disease states ( 12,13,(15)(16)(17)(18)(19). This diversity is regulated by differences in cardiolipin biosynthesis and catabolism, remodeling acyl selectivities, and transacylase as well as acyltransferase activities ( 20,21 ).…”

“…Expansion of this acyl fragment region shows the presence of multiple very low abundance fatty acyl Mouse hepatic mitochondrial lipids were extracted by a modifi ed Bligh and Dyer procedure as described in the section of "Materials and Methods." The cardiolipin molecular species in the lipid extracts were identifi ed and quantifi ed as previously described ( 15 ). The mass content and distribution of cardiolipin species represent the mean of four separately prepared mitochondria from different animals.…”