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Garcia-Villalba R.,University of Granada | Carrasco-Pancorbo A.,University of Granada | Nevedomskaya E.,Biomolecular Mass Spectrometry Unit | Mayboroda O.A.,Biomolecular Mass Spectrometry Unit | And 3 more authors.
Analytical and Bioanalytical Chemistry | Year: 2010

Olive oil polyphenols have important biological properties which closely depend on their bioavailability; it is, therefore, essential to understand how polyphenols are absorbed, metabolized, and eliminated from the body. An analytical method based on rapid-resolution liquid chromatography (RRLC) coupled with mass spectrometric detection with a time-of-flight analyzer (RRLC-ESI-TOF MS) has been developed for analysis of the main olive oil phenolic compounds and their metabolites in human urine. Urine samples from ten healthy volunteers were collected before and 2, 4, and 6 h after intake of 50 mL extra-virgin olive oil. The proposed method includes liquid-liquid extraction with ethyl acetate, which provides extraction recoveries of the phenolic compounds studied between 35 and 75% from spiked urine samples. Good repeatability was obtained-the relative standard deviations (RSDs) of peak areas in intra-day and inter-day studies were 4.3 and 6.5%, respectively. Statistical studies enabled us to discriminate between urine samples before and after intake, and facilitated the search for m/z values enabling this discrimination. Based on the very accurate mass information and the isotopic pattern provided by the TOF MS analyzer, together with other available information, ten of these biomarkers and more than 50 metabolites, obtained through phase I and phase II biotransformation reactions, were tentatively identified. Additionally, kinetic studies were conducted on the metabolites identified as possible biomarkers; for most of the compounds concentrations were maximum in the first two hours. © 2010 Springer-Verlag.


Hurtado-Fernandez E.,University of Granada | Pacchiarotta T.,Biomolecular Mass Spectrometry Unit | Gomez-Romero M.,University of Granada | Schoenmaker B.,Biomolecular Mass Spectrometry Unit | And 5 more authors.
Journal of Chromatography A | Year: 2011

We have developed an analytical method using UHPLC-UV/ESI-TOF MS for the comprehensive profiling of the metabolites found in the methanolic extracts of 13 different varieties of avocado at two different ripening degrees. Both chromatographic and detection parameters were optimized in order to maximize the number of compounds detected and the sensitivity. After achieving the optimum conditions, we performed a complete analytical validation of the method with respect to its linearity, sensitivity, precision, accuracy and possible matrix effects. The LODs ranged from 1.64 to 730.54. ppb (in negative polarity) for benzoic acid and chrysin, respectively, whilst they were found within the range from 0.51 to 310.23. ppb in positive polarity. The RSDs for repeatability test did not exceed 7.01% and the accuracy ranged from 97.2% to 102.0%. Our method was then applied to the analysis of real avocado samples and advanced data processing and multivariate statistical analysis (PCA, PLS-DA) were carried out to discriminate/classify the examined avocado varieties. About 200 compounds belonging to various structural classes were tentatively identified; we are certain about the identity of around 60 compounds, 20 of which have been quantified in terms of their own commercially available standard. © 2011 Elsevier B.V.


Garcia-Villalba R.,University of Granada | Pacchiarotta T.,Biomolecular Mass Spectrometry Unit | Carrasco-Pancorbo A.,University of Granada | Segura-Carretero A.,University of Granada | And 3 more authors.
Journal of Chromatography A | Year: 2011

A new analytical approach based on gas chromatography coupled to atmospheric pressure chemical ionization-time of flight mass spectrometry was evaluated for its applicability for the analysis of phenolic compounds from extra-virgin olive oil. Both chromatographic and MS parameters were optimized in order to improve the sensitivity and to maximize the number of phenolic compounds detected. We performed a complete analytical validation of the method with respect to its linearity, sensitivity, precision, accuracy and possible matrix effects. The LODs ranged from 0.13 to 1.05 ppm for the different tested compounds depending on their properties. The RSDs for repeatability test did not exceed 6.07% and the accuracy ranged from 95.4% to 101.5%. To demonstrate the feasibility of our method for analysis of real samples, we analyzed the extracts of three different commercial extra-virgin olive oils. We have identified unequivocally a number of phenolic compounds and obtained quantitative information for 21 of them. In general, our results show that GC-APCI-TOF MS is a flexible platform which can be considered as an interesting tool for screening, structural assignment and quantitative determination of phenolic compounds from virgin olive oil. © 2011 Elsevier B.V.


Ramautar R.,University Utrecht | Ramautar R.,Biomolecular Mass Spectrometry Unit | Nevedomskaya E.,Biomolecular Mass Spectrometry Unit | Mayboroda O.A.,Biomolecular Mass Spectrometry Unit | And 6 more authors.
Molecular BioSystems | Year: 2011

The potential of capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) using capillaries coated with a triple layer of polybrene-dextran sulfate-polybrene (PB-DS-PB) was evaluated for metabolic profiling of human urine. The method covers various metabolite classes and stable metabolic profiles of urine samples were obtained with favourable migration time repeatability (RSDs < 1%). The PB-DS-PB CE-TOF-MS method was used for the analysis of human urine samples from 30 males and 30 females, which had been previously analyzed by reversed-phase UPLC-TOF-MS. Multivariate data analysis of the obtained data provided clear distinction between urine samples from males and females, emphasizing gender differences in metabolic signatures. Nearly all compounds responsible for male-female classification in CE-TOF-MS were different from the classifying compounds in UPLC-TOF-MS. Almost all compounds causing classification in the CE-TOF-MS study were highly polar and did not exhibit retention in the reversed-phase UPLC system. In addition, the CE-TOF-MS classifiers had an m/z value in the range of 50-150, whereas 95% of the classifying features found with UPLC-TOF-MS had an m/z value above 150. The CE-TOF-MS method therefore appears to be highly complementary to the UPLC-TOF-MS method providing classification based on different classes of metabolites. © The Royal Society of Chemistry 2011.


Giera M.,VU University Amsterdam | Kloos D.-P.,VU University Amsterdam | Kloos D.-P.,Biomolecular Mass Spectrometry Unit | Raaphorst A.,VU University Amsterdam | And 4 more authors.
Analyst | Year: 2011

Malondialdehyde (MDA) has become a well-established biomarker for oxidative stress. The most commonly used way to determine urinary MDA levels is the thiobarbituric acid (TBA) assay, which suffers from several drawbacks. In this manuscript, we describe a novel derivatization strategy for the highly sensitive and selective fluorescence-based determination of MDA in urinary samples. The methodology is based on the mild labeling of MDA with 2-aminoacridone, which can be carried out in aqueous citrate buffer at 40°C, yielding a highly fluorescent substance. No further sample preparation than mixing with the necessary chemicals is necessary. The formed MDA derivative can conveniently be separated from the label itself and matrix constituents by gradient LC in less than 5 minutes on a cyano-based reversed-phase material. The method was validated with respect to matrix effects, linearity, selectivity and sensitivity (values as low as 1.8 nM for the LOD and 5.8 nM for the LOQ could be achieved). Standard addition quantitation was applied for the determination of MDA in human urine samples. Additionally, the protocol was applied to the measurement of a stability indicating analysis of MDA in urine at different storage conditions. © 2011 The Royal Society of Chemistry.


Lonardi E.,Biomolecular Mass Spectrometry Unit | Balog C.I.,Biomolecular Mass Spectrometry Unit | Deelder A.M.,Biomolecular Mass Spectrometry Unit | Wuhrer M.,Biomolecular Mass Spectrometry Unit
Expert Review of Proteomics | Year: 2010

Glycan microarrays are emerging as increasingly used screening tools with a high potential for unraveling protein-carbohydrate interactions: probing hundreds or even thousands of glycans in parallel, they provide the researcher with a vast amount of data in a short time-frame, while using relatively small amounts of analytes. Natural glycan microarrays focus on the glycans repertoire of natural sources, including both well-defined structures as well as still-unknown ones. This article compares different natural glycan microarray strategies. Glycan probes may comprise oligosaccharides from glycoproteins as well as glycolipids and polysaccharides. Oligosaccharides may be purified from scarce biological samples that are of particular relevance for the carbohydrate-binding protein to be studied. We give an overview of strategies for glycan isolation, derivatization, fractionation, immobilization and structural characterization. Detection methods such as fluorescence analysis and surface plasmon resonance are summarized. The importance of glycan density and multivalency is discussed. Furthermore, some applications of natural glycan microarrays for studying lectin and antibody binding are presented. © 2010 Expert Reviews Ltd.


Kloos D.,VU University Amsterdam | Kloos D.,Biomolecular Mass Spectrometry Unit | Derks R.J.E.,Biomolecular Mass Spectrometry Unit | Wijtmans M.,VU University Amsterdam | And 6 more authors.
Journal of Chromatography A | Year: 2012

The analysis of cellular metabolic processes is of fundamental biological interest. Cellular metabolites, such as the intermediates of the tricarboxylic acid (TCA) cycle, provide essential information about the metabolic state of the cell. Not only is the TCA cycle a key factor in the energy regulation within aerobic cells, it possibly also plays a role in cell signaling. This paper describes a novel derivatization strategy, using the empirically selected N-methyl-2-phenylethanamine as derivatization reagent with a carbodiimide as co-reagent, for the selective derivatization of carboxylic acids, such as the di- and tri-carboxylic acids of the TCA cycle. Optimization of the derivatization protocol is described. This procedure enables analysis of the derivatives using on-line solid-phase extraction and reversed-phase liquid chromatography in combination with sensitive positive-ion electrospray ionization mass spectrometry. The complete procedure, involving the use of core-shell silica column material, allows the rapid analysis of TCA cycle intermediates in sample matrices, here shown for pig heart tissue extracts, with a good linearity over 3-4 orders of magnitude. Detection limits range from 12 to 1000. nM, depending on the analyte. © 2011 Elsevier B.V.


Pacchiarotta T.,Biomolecular Mass Spectrometry Unit | Nevedomskaya E.,Biomolecular Mass Spectrometry Unit | Carrasco-Pancorbo A.,Biomolecular Mass Spectrometry Unit | Deelder A.M.,Biomolecular Mass Spectrometry Unit | Mayboroda O.A.,Biomolecular Mass Spectrometry Unit
Journal of Biomolecular Techniques | Year: 2010

With a development of the metabolomics field, complementary cross-platform approaches started to attract attention, as none of the contemporary analytical methods had the capacity to cover the entire space of the human metabolome. In the current manuscript, we have evaluated an online coupling of gas chromatography (GC)-mass spectrometry (MS) and flame ionization detector (FID) as ways of cross-detector analysis. The possible value of this combination was recognized from the very first days of GC-MS history but was never explored in detail. We have compared the basic analytical parameters of both detectors, such as limit of detection (LOD) and limit of quantification, with intra- and interday reproducibility. We show that for the majority of the tested compounds, MS detector demonstrates lower LOD. At the same time, FID appeared to be more robust, showing lower relative standard deviations (RSDs) for intra- and interday reproducibility. We conclude that the gain of this dual detector acquisition appears to be most evident for complex biological samples, where wide dynamic range and predictable response of FID are useful for an initial quantitative overview of sample composition and estimation of molar proportions of different metabolites. MS provides reliable, structural information and superior, at least in the case of atmospheric pressure chemical ionization, sensitivity. Taken together, both detectors represent a flexible tool for explorative studies and if supported by a powerful data-processing algorithm, would appear to be useful in any metabolic profiling study. © 2010 ABRF.


Schimmel J.,LUMC | Balog C.I.A.,Biomolecular Mass Spectrometry Unit | Deelder A.M.,Biomolecular Mass Spectrometry Unit | Drijfhout J.W.,LUMC | And 2 more authors.
Journal of Proteomics | Year: 2010

Covalent attachment of Small Ubiquitin-like MOdifiers (SUMOs) to the ε-amino group of lysine residues in target proteins regulates many cellular processes. Previously, we have identified the 110 kDa U4/U6.U5 tri-snRNP component SART1 as a target protein for SUMO-1 and SUMO-2. SART1 contains lysines on positions 94, 141, 709 and 742 that are situated in tetrameric sumoylation consensus sites. Recombinant SART1 was produced in E. coli, conjugated to SUMO-2 in vitro, digested by trypsin and analysed by MALDI-ToF, MALDI-FT-ICR or nanoLC-iontrap MS/MS. We found that Lys94 and Lys141 of SART1 were preferentially conjugated to SUMO-2 monomers and multimers in vitro. In agreement with these results, mutation of Lys94 and Lys141, but not Lys709 and Lys742, resulted in a reduced sumoylation of SART1 in HeLa cells. A detailed characterization of the four sumoylation sites of SART1 using full-length recombinant SART1 and a peptide sumoylation approach indicated that positively charged amino acids adjacent to the tetrameric sumoylation consensus site enhance the sumoylation of Lys94. These results show that amino acids surrounding the classic tetrameric SUMO consensus site can regulate sumoylation efficiency and validate the use of an in vitro sumoylation-mass spectrometry approach for the identification of sumoylation sites. © 2010 Elsevier B.V. All rights reserved.


Palmblad M.,Biomolecular Mass Spectrometry Unit | Drijfhout J.W.,Leiden University | Deelder A.M.,Biomolecular Mass Spectrometry Unit
Journal of Combinatorial Chemistry | Year: 2010

Combinatorial libraries can be characterized in detail by high resolving power mass spectrometry. We here demonstrate this for synthetic decapeptide libraries using a state-of-the-art 15 T Fourier transform ion cyclotron resonance mass spectrometer. Using comparison between predicted and measured spectra, a number of metrics can be derived that shed light on the library composition and degeneracy of elemental compositions. These techniques can be used to rapidly quality control combinatorial synthesis products or follow combinatorial, libraries in more detail, for instance during binding studies or chemical reactions. The methods are in principle equally applicable to the analysis of other types of combinatorial libraries. © 2010 American Chemical Society.

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