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Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem mass spectrometry with electrospray ionization.

ABSTRACT: We describe a simple tandem mass spectrometric approach toward structural characterization of mycolic acids, the long-chain ?-alkyl-?-hydroxy fatty acids unique to mycobacteria and related taxa. On collisionally activated dissociation in a linear ion trap or tandem quadrupole mass spectrometer, the [M-H](-) ions of mycolic acid generated by electrospray ionization undergo dissociation to eliminate the meroaldehyde residue, leading to formation of carboxylate anions containing ?-alkyl chains. The structural information from these fragment ions affords structural assignment of the mycolic acids, including the lengths of the meromycolate chain and the ?-branch. This study revealed that the mycolic acids isolated from pathogenic Rhodococcus equi 103 contained a series of homologous ions having C(30) to C(50) chain with 0-2 double bonds. The ?-branch ranged from C(10) to C(18) with 0 to 1 double bond, in which 16:0 and 14:0 are the most prominent, whereas the meromycolate chain ranged from C(14) to C(34) with 0 to 2 double bonds. The major molecular species consisted of more than 3 isomers that differ by the lengths of the ?-branch or meromycolate chain, and up to 10 isobaric isomers were identified for some minor ions. We also employed tandem quadrupole mass spectrometry with precursor ion and neutral loss scans for profiling mycolic acid with specific structure in mixtures. The tandem spectra obtained from precursor ion scans of m/z 255 (16:0-carboxylate anion) and m/z 227 (14:0-carboxylate anion) may provide a simple specific means for classification of Rhodococci species, whereas tandem spectra from neutral loss of meroaldehyde residue scans provided a simple approach to reveal the mycolic acid molecules with specific meromycolate chain in mixtures.

SUBMITTER: Hsu FF

PROVIDER: S-EPMC3006067 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem mass spectrometry with electrospray ionization.

Hsu Fong-Fu FF Soehl Kristina K Turk John J Haas Albert A

Analytical biochemistry 20101012 1

We describe a simple tandem mass spectrometric approach toward structural characterization of mycolic acids, the long-chain α-alkyl-β-hydroxy fatty acids unique to mycobacteria and related taxa. On collisionally activated dissociation in a linear ion trap or tandem quadrupole mass spectrometer, the [M-H](-) ions of mycolic acid generated by electrospray ionization undergo dissociation to eliminate the meroaldehyde residue, leading to formation of carboxylate anions containing α-alkyl chains. The ...[more]

PMID: 20946862

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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.

Dataset Information

Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem mass spectrometry with electrospray ionization.

Publications

Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem mass spectrometry with electrospray ionization.

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