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Development of MALDI-TOF methodology for selective MS-analysis and imaging of cardiolipins in lipid extracts and tissues.

Sparvero-LJ; Amoscato-A; Pitt-BR; Bayir-H; Kagan-VE
Toxicologist 2011 Mar; 120(Suppl 2):275
Cardiolipin (CL) is an anionic phospholipid that plays a key role in mitochondrial electron transport but is also highly susceptible to oxidative stress. The molecular diversity of endogenous cardiolipins in some tissues and the low abundance of its individual species, particularly oxidized derivatives, makes in situ analysis and imaging by MALDI-Mass Spectrometry (MALDI-MS) challenging. The goal of this study was to develop a method to selectively analyze CL using existing CL probes in a novel way, to decrease CL mass spectral complexity. Nonyl Acridine Orange (NAO) is a commonly used fluorescent stain for cardiolipin. We have used NAO as a novel MALDI matrix, and have found that it allowed detection of anionic phospholipids, including cardiolipin in the negative ion mode. With careful laser tuning, NAO induced prompt decay of cardiolipin selectively - but not other phospholipids - at very high efficiencies. From these selective prompt decay products, much information about the structure of cardiolipin can be determined. Pseudo- MS3 is possible on a MALDI-TOF instrument by doing MS/MS on these products of cardiolipin. We have also extended these findings to include oxidized species of CL's and have determined that NAO also induced prompt decay of oxidized CL. Prompt fragments are seldom seen with dihydroxy-benzoic acid (DHB) which has been employed as a standard matrix for lipid analysis in the negative ion mode. Our results indicate that NAO can be utilized as a discerning matrix for the assessment of CL in lipid extracts from cells and the distribution of CL - particularly in tissues with a high diversity of CL molecular species and their oxidation products - by taking full advantage of the unique ability to induce prompt decay of this phospholipid with NAO.
Biological-effects; Cell-biology; Cell-function; Cell-morphology; Cell-transformation; Cellular-reactions; Inhalation-studies; Medical-research; Oxidation; Oxidative-processes; Physical-properties; Physiological-effects; Risk-analysis; Risk-factors; Statistical-analysis; Mass-spectrometry; Phospholipids; Diagnostic-techniques; Tissue-culture; Toxicology
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The Toxicologist. Society of Toxicology 50th Annual Meeting and ToxExpo, March 6-10, 2011, Washington, DC
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University of Pittsburgh at Pittsburgh