Heiko Hayen

Proteomics characterization of mouse kidney peroxisomes by tandem mass spectrometry and protein correlation profiling.

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by α- and β-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate ∼50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.

Strategies for the liquid chromatographic-mass spectrometric analysis of non-polar compounds

Electrospray ionization and atmospheric pressure chemical ionization (APCI) have evolved recently as very useful tools for the liquid chromatographic-mass spectrometric (LC-MS) analysis of polar substances. Non-polar compounds, however, are difficult to analyze with these atmospheric pressure ionization techniques due to their soft ionization mechanism. Recently, new approaches have been introduced which are likely to overcome this obstacle, at least partly. On-line electrochemical conversion of the analytes to more polar reaction products, atmospheric pressure photoionization, atmospheric pressure electron capture negativeion-MS and coordination ionspray-MS are four techniques which are presented in detail compared and discussed critically with respect to their current status and future perspectives. Particular focus is directed from a chemical viewpoint on the substance groups which are accessible by each of the new approaches.

Iron Acquisition by Phytosiderophores Contributes to Cadmium Tolerance

Based on the ability of phytosiderophores to chelate other heavy metals besides iron (Fe), phytosiderophores were suggested to prevent graminaceous plants from cadmium (Cd) toxicity. To assess interactions between Cd and phytosiderophore-mediated Fe acquisition, maize (Zea mays) plants were grown hydroponically under limiting Fe supply. Exposure to Cd decreased uptake rates of 59Fe(III)-phytosiderophores and enhanced the expression of the Fe-phytosiderophore transporter gene ZmYS1 in roots as well as the release of the phytosiderophore 2′-deoxymugineic acid (DMA) from roots under Fe deficiency. However, DMA hardly mobilized Cd from soil or from a Cd-loaded resin in comparison to the synthetic chelators diaminetriaminepentaacetic acid and HEDTA. While nano-electrospray-high resolution mass spectrometry revealed the formation of an intact Cd(II)-DMA complex in aqueous solutions, competition studies with Fe(III) and zinc(II) showed that the formed Cd(II)-DMA complex was weak. Unlike HEDTA, DMA did not protect yeast (Saccharomyces cerevisiae) cells from Cd toxicity but improved yeast growth in the presence of Cd when yeast cells expressed ZmYS1. When supplied with Fe-DMA as a Fe source, transgenic Arabidopsis (Arabidopsis thaliana) plants expressing a cauliflower mosaic virus 35S-ZmYS1 gene construct showed less growth depression than wild-type plants in response to Cd. These results indicate that inhibition of ZmYS1-mediated Fe-DMA transport by Cd is not related to Cd-DMA complex formation and that Cd-induced phytosiderophore release cannot protect maize plants from Cd toxicity. Instead, phytosiderophore-mediated Fe acquisition can improve Fe uptake in the presence of Cd and thereby provides an advantage under Cd stress relative to Fe acquisition via ferrous Fe.

Dielectric barrier discharge ionization for liquid chromatography/mass spectrometry.

An atmospheric pressure microplasma ionization source based on a dielectric barrier discharge with a helium plasma cone outside the electrode region has been developed for liquid chromatography/mass spectrometry (LC/MS). For this purpose, the plasma was realized in a commercial atmospheric pressure ionization source. Dielectric barrier discharge ionization (DBDI) was compared to conventional electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) in the positive ionization mode. Therefore, a heterogeneous compound library was investigated that covered polar compounds such as amino acids, water-soluble vitamins, and nonpolar compounds like polycyclic aromatic hydrocarbons and functionalized hydrocarbons. It turned out that DBDI can be regarded as a soft ionization technique characterized by only minor fragmentation similar to APCI. Mainly protonated molecules were detected. Additionally, molecular ions were observed for polycyclic aromatic hydrocarbons and derivatives thereof. During DBDI, adduct formation with acetonitrile occurred. For aromatic compounds, addition of one to four oxygen atoms and to a smaller extend one nitrogen and oxygen was observed which delivered insight into the complexity of the ionization processes. In general, compounds covering a wider range of polarities can be ionized by DBDI than by ESI. Furthermore, limits of detection compared to APCI are in most cases equal or even better.

Labelling of proteins with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and lanthanides and detection by ICP-MS

The labelling of two different proteins (bovine serum albumin, hen egg white lysozyme) with the commercially available chelating compound 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA) has been investigated. The assay described here has been optimised for the application to detect proteins labelled by stable isotopes of Eu, Tb and Ho and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Detection has been performed by laser ablation ICP-MS after electroblotting of the target proteins onto NC membranes. A range of total protein amounts from 0.015 pmol (BSA) to 105 pmol (lysozyme) has been covered. A calibration was performed for BSA in the range from 0.015 to 15 pmol and a limit of detection below 15 fmol can be estimated. For lysozyme integrated sensitivities of more than 107 cps pmol−1 of protein have been realized. The conditions, once optimised for labelling with Eu, have been applied for other lanthanides (Tb, Ho), too. ESI-MS of the intact and the tryptic digested lysozyme has been used as to provide a better understanding of the reaction chemistry and efficiency. The procedure described here looks promising to develop multielement labelling strategies (multiplexing) for ICP-MS applications in quantitative proteomics.

In vivo and in vitro degradation of poly(ether ester) block copolymers based on poly(ethylene glycol) and poly(butylene terephthalate).

Two in vivo degradation studies were performed on segmented poly(ether ester)s based on polyethylene glycol (PEG) and poly(butylene terephthalate) (PBT) (PEOT/PBT). In a first series of experiments, the in vivo degradation of melt-pressed discs of different copolymer compositions were followed up for 24 weeks after subcutaneous implantation in rats. The second series of experiments aimed to simulate long-term in vivo degradation. For this, PEOT/PBT samples were pre-degraded in phosphate buffer saline (PBS) at 100 degrees C and subsequently implanted. In both series, explanted materials were characterized by intrinsic viscosity measurements, mass loss, proton nuclear magnetic resonance spectroscopy (1H-NMR) and differential scanning calorimetry (DSC). In both studies the copolymer with the higher PEO content degraded the fastest, although all materials degraded relatively slowly. To determine the nature of the degradation products formed during hydrolysis of the copolymers, 1000 PEOT71PBT29 (a copolymer based on PEG with a molecular weight of 1000 g/mol and 71 wt% of PEO-containing soft segments) was degraded in vitro at 100 degrees C in phosphate buffer saline (PBS) during 14 days. The degradation products present in PBS were analyzed by 1H-NMR and high performance liquid chromatography/mass spectroscopy (HPLC/MS). These degradation products consisted of a fraction with high contents of PEO that was soluble in PBS and a PEOT/PBT fraction that was insoluble at room temperature. From the different in vitro and in vivo degradation experiments performed, it can be concluded that PEOT/PBT degradation is a slow process and generates insoluble polymeric residues with high PBT contents.

Covalent protein modification by reactive drug metabolites using online electrochemistry/liquid chromatography/mass spectrometry.

We present a rapid and convenient method to perform and evaluate the covalent protein binding of reactive phase I metabolites. The oxidative metabolism of the drugs paracetamol, amodiaquine, and clozapine is simulated in an electrochemical (EC) flow-through cell, which is coupled online to an LC/MS system. Adduct formation of the reactive metabolites with the proteins beta-lactoglobulin A and human serum albumin proceeds in a reaction coil between EC cell and injection system of the HPLC system. The formed drug-protein adducts are characterized with online time-of-flight mass spectrometry, and the modification site is localized using FTICR-mass spectrometry. Due to its simple setup, easy handling, and short analysis times, the method provides an interesting tool for the rapid risk assessment of covalent protein binding as well as for the synthesis of covalent drug-protein adducts in high purity and high yield.

Characterization of oligomeric compounds in secondary organic aerosol using liquid chromatography coupled to electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The components of secondary organic aerosols (SOAs) generated from the gas-phase ozonolysis of two C(10)H(16)-terpenes (alpha-pinene; sabinene) and a cyclic C(6)H(10) alkene (cyclohexene) were characterized by the use of a Fourier transform ion cyclotron mass spectrometer equipped with an electrospray ionization source operated in the negative ion mode. Reversed-phase high-performance liquid chromatography was used to achieve chromatographic separation of highly oxidized organic compounds. In addition to the well-known group of low molecular weight oxidation products (monomers; e.g. dicarboxylic acids), higher molecular weight compounds (dimers) were also detected and their exact elemental compositions were determined. In order to provide additional information for the structural elucidation of these compounds, collision-induced dissociation was applied. Based on the MS/MS spectra, two higher molecular weight products are proposed to be an ester and a peroxide. Molecular formulae calculated from the exact masses show that the SOA-compounds are heavily oxidized and this information creates the background to a discussion of potential reaction pathways for the formation of higher molecular weight compounds.

Labelling of proteins by use of iodination and detection by ICP-MS

The labelling of three different proteins, bovine serum albumin (BSA), chicken egg white lysozyme and porcine gastric mucosa pepsin, with iodine using a commercially available reaction kit has been investigated for total protein amounts of 500 μg. The assay described here has already often been applied but with radioactive iodine isotopes and is extended in this work for the use of the stable isotope 127I and detection by ICP-MS. The reaction conditions are investigated and optimised for proteins separated by SDS-PAGE and electroblotted onto NC membranes. Detection has been performed by laser ablation ICP-MS on the membranes. Long reaction times result in degradation of proteins, whereas for short reaction times (4 min) integrated sensitivities of even 108 cps pmol−1 of protein have been realized. Total amounts of proteins have been investigated in the range from 0.015 (BSA) to 105 pmol (lysozyme). A calibration was performed for BSA from 0.015 to 15 pmol but the limit of detection was reached already at about 150 fmol due to high iodine blank values, only. The labelling procedure described here does not affect protein mobility in SDS-PAGE, because the change of the molecular weight is very moderate for the proteins investigated here. This is quite opposite to those procedures where chelating compounds are applied. ESI-FTICR-MS has been used to identify histidine and tyrosine as the target amino acids for iodination. Additionally, oxidation of methionine residues has been observed.

LC-Electron capture APCI-MS for the determination of nitroaromatic compounds

The determination of selected nitroaromatic compounds in liquid chromatography-mass spectrometry with electron capture (EC) ionisation using a commercial atmospheric pressure chemical ionisation (APCI) interface in the negative mode is described. The electron capture effect is observed for nitroaromatics which do not easily undergo deprotonation under these conditions. Depending on the structure of the analytes, either dissociative or, for the first time in LC-MS, non-dissociative electron capture is observed. Limits of detection and linear range for the determination of the analytes match those obtained for nitroaromatics which undergo deprotonation. The investigated substances comprise numerous substituted nitrobenzenes and nitrobenzooxadiazoles.