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Electrospray ionization tandem mass spectrometry of sodiated adducts of cholesteryl esters.

ABSTRACT: Cholesteryl esters (CE) are important lipid storage molecules. The present study demonstrates that sodiated adducts of CE molecular species form positive ions that can be detected in both survey scan mode as well as by exploiting class-specific fragmentation in MS/MS scan modes. A common neutral loss for CE is the loss of cholestane (NL 368.5), which can be used to specifically quantify tissue CE molecular species. Using this MS/MS technique, CE molecular species were quantified in mouse monocyte-derived macrophages (J774 cells) incubated with either linoleic (18:2) or arachidonic acid (20:4). These studies revealed that arachidonic acid was not only incorporated into the CE pool, but also was elongated resulting in the accumulation of 22:4 and 24:4 CE molecular species in macrophages. Additionally, this technique was used to quantify CE molecular species present in crude lipid extracts from plasma of female mice fed a Western diet, which led to an enrichment in CE molecular species containing monounsaturated fatty acids compared to female mice fed a normal chow diet. Last, NL 368.5 spectra revealed the oxidation of the aliphatic fatty acid residues of CE molecular species containing polyunsaturated fatty acids. Taken together, these studies demonstrate the utility of using sodiated adducts of CE in conjunction with direct infusion electrospray ionization tandem mass spectrometry to rapidly quantify CE molecular species in biological samples.

SUBMITTER: Bowden JA

PROVIDER: S-EPMC3215890 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Electrospray ionization tandem mass spectrometry of sodiated adducts of cholesteryl esters.

Bowden John A JA Shao Fei F Albert Carolyn J CJ Lally John W JW Brown Robert J RJ Procknow Jesse D JD Stephenson Alan H AH Ford David A DA

Lipids 20110909 12

Cholesteryl esters (CE) are important lipid storage molecules. The present study demonstrates that sodiated adducts of CE molecular species form positive ions that can be detected in both survey scan mode as well as by exploiting class-specific fragmentation in MS/MS scan modes. A common neutral loss for CE is the loss of cholestane (NL 368.5), which can be used to specifically quantify tissue CE molecular species. Using this MS/MS technique, CE molecular species were quantified in mouse monocyt ...[more]

PMID: 21904795

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Project description:BackgroundWax esters are highly hydrophobic neutral lipids that are major constituents of the cutin and suberin layer. Moreover they have favorable properties as a commodity for industrial applications. Through transgenic expression of wax ester biosynthetic genes in oilseed crops, it is possible to achieve high level accumulation of defined wax ester compositions within the seed oil to provide a sustainable source for such high value lipids. The fatty alcohol moiety of the wax esters is formed from plant-endogenous acyl-CoAs by the action of fatty acyl reductases (FAR). In a second step the fatty alcohol is condensed with acyl-CoA by a wax synthase (WS) to form a wax ester. In order to evaluate the specificity of wax ester biosynthesis, analytical methods are needed that provide detailed wax ester profiles from complex lipid extracts.ResultsWe present a direct infusion ESI-tandem MS method that allows the semi-quantitative determination of wax ester compositions from complex lipid mixtures covering 784 even chain molecular species. The definition of calibration prototype groups that combine wax esters according to their fragmentation behavior enables fast quantitative analysis by applying multiple reaction monitoring. This provides a tool to analyze wax layer composition or determine whether seeds accumulate a desired wax ester profile. Besides the profiling method, we provide general information on wax ester analysis by the systematic definition of wax ester prototypes according to their collision-induced dissociation spectra. We applied the developed method for wax ester profiling of the well characterized jojoba seed oil and compared the profile with wax ester-accumulating Arabidopsis thaliana expressing the wax ester biosynthetic genes MaFAR and ScWS.ConclusionsWe developed a fast profiling method for wax ester analysis on the molecular species level. This method is suitable to screen large numbers of transgenic plants as well as other wax ester samples like cuticular lipid extracts to gain an overview on the molecular species composition. We confirm previous results from APCI-MS and GC-MS analysis, which showed that fragmentation patterns are highly dependent on the double bond distribution between the fatty alcohol and the fatty acid part of the wax ester.

Project description:The tobacco-specific nitrosamines N'-nitrosonornicotine (NNN, 1) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 2) are potent carcinogens in rodents. Bioactivation of NNN and NNK by cytochrome P450 enzymes generates a pyridyloxobutylating agent 6, which alkylates DNA to produce pyridyloxobutyl (POB)-DNA adducts. POB-DNA adduct formation plays a critical role in NNN and NNK carcinogenicity in rodents. To further investigate the significance of this pathway, we developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantitative analysis of four POB-DNA adducts with known structures. The corresponding deuterated analogues were synthesized and used as internal standards. DNA samples, spiked with internal standards, were subjected to neutral thermal hydrolysis followed by enzymatic hydrolysis. The hydrolysates were partially purified by solid phase extraction prior to HPLC-ESI-MS/MS analysis. The method was accurate and precise. Excellent sensitivity was achieved, especially for O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd, 11) with a detection limit of 100 amol per mg DNA. DNA samples treated with different concentrations of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 3) were subjected to HPLC-ESI-MS/MS analysis. 7-[4-(3-Pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 12) was the most abundant adduct, followed by O6-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O6-POB-dGuo, 8), O2-POB-dThd, and O2-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (O2-POB-Cyt, 13). Lung and liver DNA isolated from NNK-treated rats were analyzed. Consistent with the in vitro data, 7-POB-Gua was the major POB-DNA adduct formed in vivo. However, levels of O6-POB-dGuo were the lowest of the four adducts analyzed, suggesting efficient repair of this adduct in vivo. In contrast to the other three adducts, O6-POB-dGuo was more abundant in lung than in liver. O2-POB-dThd appeared to be poorly repaired in vivo, and its levels were comparable to those of 7-POB-Gua. The results of this study provide a sensitive HPLC-ESI-MS/MS method for comprehensive quantitation of four POB-DNA adducts, support an important role of O6-POB-dGuo in NNK lung tumorigenicity in rats, and suggest that O2-POB-dThd may be a useful tobacco-specific DNA biomarker for future tobacco carcinogenesis studies.

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Electrospray ionization tandem mass spectrometry of sodiated adducts of cholesteryl esters.

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Electrospray ionization tandem mass spectrometry of sodiated adducts of cholesteryl esters.

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