A matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) based approach was developed for the rapid analyses of cellular glycerophospholipids. Through multiplexed solvent-enabled optimization of analyte-matrix interactions during the crystallization process, over a 30-fold increase in S/N was achieved using 9-aminoacridine as matrix. The linearity of response (r 2 =0.99) and dynamic range of this method (over 2 orders of magnitude) were excellent. Moreover, through multiplexing ionization conditions by generating suites of different analytematrix interactions in the absence or presence of different alkali metal cations in the matrix, discrete lipid classes were highly and selectively ionized under different conditions resulting in the de facto resolution of lipid classes without chromatography. The resultant decreases in spectral complexity facilitated tandem mass spectrometric analysis through high energy fragmentation of lithiated molecular ions that typically resulted in informative fragment ions. Anionic phospholipids were also detected as singly negatively charged species that could be fragmented using MALDI tandem mass spectrometry leading to structural assignments. Collectively, these results identify a rapid, sensitive and highly informative MALDI-TOF MS approach for analysis of cellular glycerophospholipids directly from extracts of mammalian tissues without the need for prior chromatographic separation.

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Genetic ablation of calcium-independent phospholipase

Large-scale neuronal remodeling through apoptosis occurs shortly after birth in all known mammalian species. Apoptosis, in large part, depends upon critical interactions between mitochondrial membranes and cytochrome c. Herein, we examined the hypothesis that the largescale reorganization of neuronal circuitry after birth is accompanied by profound alterations in cardiolipin (CL) content and molecular species distribution. During embryonic development, over 100 CL molecular species were identified and quantitated in murine neuronal tissues. The embryonic CL profile was notable for the presence of abundant amounts of relatively short aliphatic chains (e.g., palmitoleic and oleic acids). In sharp contrast, after birth, the CL profile contained a remarkably complex repertoire of CL molecular species, in which the signaling fatty acids (i.e., arachidonic and docosahexaenoic acids) were markedly increased. These results identify the rapid remodeling of CL in the perinatal period with resultant alterations in the physical properties of the mitochondrial membrane. The complex distribution of aliphatic chains in the neuronal CL pool is separate and distinct from that in other organs (e.g., heart, liver, etc.), where CL molecular species contain predominantly only one major type of aliphatic chain (e.g., linoleic acid). Analyses of mRNA levels by real-time quantitative polymerase chain reactions suggested that the alterations in CL content were due to the combined effects of both attenuation of de novo CL biosynthesis and decreased remodeling of CL. Collectively, these results provide a new perspective on the complexity of CL in neuronal signaling, mitochondrial bioenergetics, and apoptosis.Cardiolipin (1,3-diphosphatidyl-sn-glycerol, CL) 1 is exclusively present in the mitochondrial membrane of eukaryotic cells (1), whose evolutionary origins can be traced back to bacterial membranes. Each CL molecular species is uniquely comprised of a dimer of two phosphatidyl † This work was supported by NIA Grants R01 AG23168 and R01 AG31675, NIH Grant P01 HL57278, and the Neurosciences Education and Research Foundation.* To whom correspondence should be addressed: Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Internal Medicine, Washington University School of Medicine, Box 8020, 660 South Euclid Avenue, St. Louis, MO 63110. Telephone: (314) 362−2690. Fax: (314) 362−1402. E-mail: xianlin@wustl.edu.. Publisher's Disclaimer: This PDF receipt will only be used as the basis for generating PubMed Central (PMC) documents. PMC documents will be made available for review after conversion (approx. 2−3 weeks time). Any corrections that need to be made will be done at that time. No materials will be released to PMC without the approval of an author. Only the PMC documents will appear on PubMed Central --this PDF Receipt will not appear on PubMed Central. 1 Abbreviation: CL, cardiolipin; FA, fatty acid or fatty acyl; FACO, fatty acyl-CoA oxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPLC, high-pe...