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ABSTRACT: An LC-MS based method for the profiling and characterization of ceramide species in the upper layer of human skin is described. Ceramide samples, collected by tape stripping of human skin, were analyzed by reversed-phase liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry operated in both positive and negative electrospray ionization mode. All known classes of ceramides could be measured in a repeatable manner. Furthermore, the data set showed several undiscovered ceramides, including a class with four hydroxyl functionalities in its sphingoid base. High-resolution MS/MS fragmentation spectra revealed that each identified ceramide species is composed of several skeletal isomers due to variation in carbon length of the respective sphingoid bases and fatty acyl building blocks. The resulting variety in skeletal isomers has not been previously demonstrated. It is estimated that over 1000 unique ceramide structures could be elucidated in human stratum corneum. Ceramide species with an even and odd number of carbon atoms in both chains were detected in all ceramide classes. Acid hydrolysis of the ceramides, followed by LC-MS analysis of the end-products, confirmed the observed distribution of both sphingoid bases and fatty acyl groups in skin ceramides. The study resulted in an accurate mass retention time library for targeted profiling of skin ceramides. It is furthermore demonstrated that targeted data processing results in an improved repeatability versus untargeted data processing (72.92% versus 62.12% of species display an RSD < 15%).
Analytical Chemistry 11/2011; 84(1):403-11. · 5.86 Impact Factor
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ABSTRACT: Comparative metabolomics of Leishmania species requires the simultaneous identification and quantification of a large number of intracellular metabolites. Here, we describe the optimisation of a comprehensive metabolite extraction protocol for Leishmania parasites and the subsequent optimisation of the analytical approach, consisting of hydrophilic interaction liquid chromatography coupled to LTQ-orbitrap mass spectrometry. The final optimised protocol starts with a rapid quenching of parasite cells to 0 °C, followed by a triplicate washing step in phosphate-buffered saline. The intracellular metabolome of 4 × 10(7) parasites is then extracted in cold chloroform/methanol/water 20/60/20 (v/v/v) for 1 h at 4 °C, resulting in both cell disruption and comprehensive metabolite dissolution. Our developed metabolomics platform can detect approximately 20% of the predicted Leishmania metabolome in a single experiment in positive and negative ionisation mode.
Analytical and Bioanalytical Chemistry 11/2010; 398(5):2059-69. · 3.78 Impact Factor
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ABSTRACT: The post-genomics era has provided researchers with access to a new generation of tools for the global characterization and understanding of pathogen diversity. This review provides a critical summary of published Leishmania post-genomic research efforts to date, and discusses the potential impact of the addition of metabolomics to the post-genomic toolbox. Metabolomics aims at understanding biology by comprehensive metabolite profiling. We present an overview of the design and interpretation of metabolomics experiments in the context of Leishmania research. Sample preparation, measurement techniques, and bioinformatics analysis of the generated complex datasets are discussed in detail. To illustrate the concepts and the expected results of metabolomics analyses, we also present an overview of comparative metabolic profiles of drug-sensitive and drug-resistant Leishmania donovani clinical isolates.
Parasitology 08/2010; 137(9):1291-302. · 2.96 Impact Factor
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ABSTRACT: Leishmaniasis is a debilitating disease caused by the parasite Leishmania. There is extensive clinical polymorphism, including variable responsiveness to treatment. We study Leishmania donovani parasites isolated from visceral leishmaniasis patients in Nepal that responded differently to antimonial treatment due to differing intrinsic drug sensitivity of the parasites. Here, we present a proof-of-principle study in which we applied a metabolomics pipeline specifically developed for L. donovani to characterize the global metabolic differences between antimonial-sensitive and antimonial-resistant L. donovani isolates. Clones of drug-sensitive and drug-resistant parasite isolates from clinical samples were cultured in vitro and harvested for metabolomics analysis. The relative abundance of 340 metabolites was determined by ZIC-HILIC chromatography coupled to LTQ-Orbitrap mass spectrometry. Our measurements cover approximately 20% of the predicted core metabolome of Leishmania and additionally detected a large number of lipids. Drug-sensitive and drug-resistant parasites showed distinct metabolic profiles, and unsupervised clustering and principal component analysis clearly distinguished the two phenotypes. For 100 metabolites, the detected intensity differed more than three-fold between the 2 phenotypes. Many of these were in specific areas of lipid metabolism, suggesting that the membrane composition of the drug-resistant parasites is extensively modified. Untargeted metabolomics has been applied on clinical Leishmania isolates to uncover major metabolic differences between drug-sensitive and drug-resistant isolates. The identified major differences provide novel insights into the mechanisms involved in resistance to antimonial drugs, and facilitate investigations using targeted approaches to unravel the key changes mediating drug resistance.
PLoS Neglected Tropical Diseases 01/2010; 4(11):e904. · 4.69 Impact Factor
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ABSTRACT: Metabolomics nowadays mostly comprises the application of both LC-MS and GC-MS based approaches. Here we investigate different extraction set-ups for these two established analytical platforms in the field of plant metabolomics. Six extraction approaches for Arabidopsis thaliana leaves, varying in extraction solvent composition, extraction temperature and order of solvent addition within the extraction sequence, were analyzed on the two platforms. Our aim was to establish the most suitable analysis protocol, practicable for both LC-MS and GC-MS analysis, in order to obtain as extensive as possible metabolome coverage. One single sample preparation procedure would save time and valuable sample while still offering the complementary datasets generated by GC-MS and LC-MS. All extraction approaches were evaluated based on the following criteria: number of detected m/z-retention time pairs, heat maps of the detected peaks, and residual enzymatic activity of invertase and phosphatase in the plant leaf extracts. Unsupervised principal component analysis (PCA) was used to evaluate grouping trends between the different extraction approaches. Quality controls, a blend of aliquots of the different extracts, were used to establish a paired evaluation of the repeatability performance of the GC-MS and LC-MS analysis. We conclude that the use of chloroform in the extraction solvent is counterproductive in an untargeted LC-MS metabolomics approach as is heating. Below room temperature (instead of heated) extraction does not significantly degrade GC-MS performance but one should be more cautious with respect to residual enzymatic activity in the plant extract.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2009; 877(29):3572-80. · 2.78 Impact Factor
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ABSTRACT: This study treats the optimization of methods for homogenizing Arabidopsis thaliana plant leaves as well as cell cultures, and extracting their metabolites for metabolomics analysis by conventional liquid chromatography electrospray ionization mass spectrometry (LC-ESI/MS). Absolute recovery, process efficiency and procedure repeatability have been compared between different pre-LC-MS homogenization/extraction procedures through the use of samples fortified before extraction with a range of representative metabolites. Hereby, the magnitude of the matrix effect observed in the ensuing LC-MS based metabolomics analysis was evaluated. Based on relative recovery and repeatability of key metabolites, comprehensiveness of extraction (number of m/z-retention time pairs) and clean-up potential of the approach (minimum matrix effects), the most appropriate sample pre-treatment was adopted. It combines liquid nitrogen homogenization for plant leaves with thermomixer based extraction using MeOH/H(2)O 80/20. As such, an efficient and highly reproducible LC-MS plant metabolomics set-up is achieved, as illustrated by the obtained results for both LC-MS (8.88%+/-5.16 versus 7.05%+/-4.45) and technical variability (12.53%+/-11.21 versus 9.31%+/-6.65) data in a comparative investigation of A. thaliana plant leaves and cell cultures, respectively.
Journal of Chromatography B 07/2008; 871(1):37-43. · 2.89 Impact Factor
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ABSTRACT: The metabolomics goal, the unbiased relative quantification of all metabolites in a biological system, still lacks a universal analytical approach. In the LC-MS line of approach, one of the major problems encountered is the polar nature of a large group of (plant) metabolites. Here, we investigate the potential of hydrophilic interaction chromatography (HILIC) and compare its qualities with extended polarity RP chromatography. Two opposite LC phase compositions (Atlantis dC18 vs. TSKgel Amide-80) are compared in a plant metabolomics setting. Both performed equally well with regard to retentive capacities, but variation in peak area was about 5% higher for the HILIC approach. Focussing on matrix effects (ME) on the other hand, it was observed that this well-known problem in RP LC-MS metabolomics was not reduced on using hydrophilic interaction chromatography.
Journal of Separation Science 06/2008; 31(9):1609-14. · 2.73 Impact Factor
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ABSTRACT: In metabolomics, major efforts are invested in the development of suitable analytical approaches. A tendency towards the use of LC-MS is nowadays very obvious. A great majority of the metabolites of interest are polar to highly polar in nature. We focus on the so-called 'extended polarity' reversed LC phases, developed specifically to allow better retention characteristics for polar compounds. Several of these phases (Atlantis dC18, Inertsil ODS-3, Zorbax XDB, Alltima HP C18) are tested for different column dimension variations (0.5, 1.0, 2.1 mm id) in a specific LC-MS metabolomics setting. Important chromatographic and mass spectrometric quality parameters such as capacity factor, separation efficiency, peak symmetry, sensitivity, and mass accuracy are taken into account. All phases show adequate retention of polar compounds and also perform well with highly aqueous mobile phase compositions. On comparing 1.0 and 2.1 mm id columns, it is clear that the potential gain in sensitivity is not achieved. Using a Lockspray device, accurate mass measurement with a Q-TOF micro is feasible within a mass range of 12 ppm if signal intensities of compound and lockmass are equated. Finally, the extended polarity RP approach in metabolomics experiments is endorsed using real plant extracts.
Journal of Separation Science 09/2007; 30(13):2002-11. · 2.73 Impact Factor
