Hubertus Irth

Biosensor discovery of thyroxine transport disrupting chemicals

Ubiquitous chemicals may interfere with the thyroid system that is essential in the development and physiology of vertebrates. We applied a surface plasmon resonance (SPR) biosensor-based screening method for the fast screening of chemicals with thyroxine (T4) transport disrupting activity. Two inhibition assays using the main thyroid hormone transport proteins, T4 binding globulin (TBG) and transthyretin (TTR), in combination with a T4-coated biosensor chip were optimized and automated for screening chemical libraries. The transport protein-based biosensor assays were rapid, high throughput and bioeffect-related. A library of 62 chemicals including the natural hormones, polychlorinated biphenyls (PCBs), polybrominated diphenylethers (PBDEs) and metabolites, halogenated bisphenol A (BPA), halogenated phenols, pharmaceuticals, pesticides and other potential environmentally relevant chemicals was tested with the two assays. We discovered ten new active compounds with moderate to high affinity for TBG with the TBG assay. Strikingly, the most potent binding was observed with hydroxylated metabolites of the brominated diphenyl ethers (BDEs) BDE 47, BDE 49 and BDE 99, that are commonly found in human plasma. The TTR assay confirmed the activity of previously identified hydroxylated metabolites of PCBs and PBDEs, halogenated BPA and genistein. These results show that the hydroxylated metabolites of the ubiquitous PBDEs not only target the T4 transport at the TTR level, but also, and to a great extent, at the TBG level where most of the T4 in humans is circulating. The optimized SPR biosensor-based transport protein assay is a suitable method for high throughput screening of large libraries for potential thyroid hormone disrupting compounds.

Development of a Selective ESI-MS Derivatization Reagent: Synthesis and Optimization for the Analysis of Aldehydes in Biological Mixtures

In LC-MS, derivatization is primarily used to improve ionization characteristics, especially for analytes that are not (efficiently) ionized by ESI or APCI such as aldehydes, sugars, and steroids. Derivatization strategies are then directed at the incorporation of a group with a permanent charge. A compound class that typically requires derivatization prior to LC-MS is the group of small aliphatic aldehydes that are, for instance, analyzed as the key biomarkers for lipid peroxidation in organisms. Here we report the development of a new tailor-made, highly sensitive, and selective derivatization agent 4-(2-(trimethylammonio)ethoxy)benzenaminium halide (4-APC) for the quantification of aldehydes in biological matrixes with positive ESI-MS/ MS without additional extraction procedures. 4-APC possesses an aniline moiety for a fast selective reaction with aliphatic aldehydes as well as a quaternary ammonium group for improved MS sensitivity. The derivatization reaction is a convenient one-pot reaction at a mild pH (5.7) and temperature (10 degrees C). As a result, an in-vial derivatization can be performed before analysis with an LC-MS/MS system. All aldehydes are derivatized within 30 min to a plateau, except malondialdehyde, which requires 300 min to reach a plateau. All derivatized aldehydes are stable for at least 35 h. Linearity was established between 10 and 500 nM and the limits of detection were in the 3-33 nM range for the aldehyde derivatives. Furthermore, the chosen design of these structures allows tandem MS to be used to monitor the typical losses of 59 and 87 from aldehyde derivatives, thereby enabling screening for aldehydes. Finally, of all aldehydes, pentanal and hexanal were detected at elevated levels in pooled healthy human urine samples.

Recent developments in protein–ligand affinity mass spectrometry

AbstractThis review provides an overview of direct and indirect technologies to screen protein–ligand interactions with mass spectrometry. These technologies have as a key feature the selection or affinity purification of ligands in mixtures prior to detection. Specific fields of interest for these technologies are metabolic profiling of bioactive metabolites, natural extract screening, and the screening of libraries for bioactives, such as parallel synthesis libraries and small combichem libraries. The review addresses the principles of each of the methods discussed, with a focus on developments in recent years, and the applicability of the methods to lead generation and development in drug discovery. FigureSchematic view of the principle of filtration based 96-well affinity selection MS binding assays

High resolution screening of plant natural product extracts for estrogen receptor alpha and beta binding activity using an online HPLC-MS biochemical detection system

A new screening technology that combines biochemical analysis with the resolution power of high-performance liquid chromatography (HPLC), referred to here as high-resolution screening (HRS) technique, is described. The capability of the HRS technology to analyze biologically active compounds in complex mixtures is demonstrated by screening a plant natural product extract library for estrogen receptor (ER) a and fi binding activity. The simultaneous structure elucidation of biologically active components in crude extracts was achieved by operating the HRS system in combination with mass spectrometry (MS). In contrast to conventional microtiter-type bioassays, the interactions of the extracts with the ER and the employed label, coumestrol, proceeded at high speed in a closed, continuous-flow reaction detection system, which was coupled directly to the outlet of a HPLC separation column. The reaction products of this homogeneous fluorescence enhancement-type assay were detected online using a flow-through fluorescence detector. Primary screening of the extract library was performed in the fast-flow injection analysis mode (FlowScreening) wherein the chromatographic separation system was bypassed. The library was screened at high speed, using two assay lines in parallel. A total of 98% of the identified hits were confirmed in a traditional 96-well microplate-based fluorescence polarization assay, indicating the reliability of the FlowScreening process. Active extracts were reassayed in a transcriptional activation assay in order to assess the functional activity of the bioactive extracts. Only functional active extracts were processed in the more time-consuming HRS mode, which was operated in combination with MS. Information on the number of active compounds, their retention times, the molecular masses, and the MS/MS-fingerprints as a function of their biological activity was obtained from 50% of the functional active extracts in real time. This dramatically enhances the speed of biologically active compound characterization in natural product extracts compared to traditional fractionation approaches.

Effect of the mobile phase composition on the separation and detection of intact proteins by reversed-phase liquid chromatography-electrospray mass spectrometry.

Various buffers (ammonium acetate, ammonium formate, and ammonium hydrogencarbonate), acids (formic acid, acetic acid, heptafluorobutyric acid, and trifluoroacetic acid), and bases (ammonium hydroxide and morpholine) covering the range from 2 to 11.5 have been investigated for their performance in the separation of proteins by reversed-phase liquid chromatography (RPLC) and in their detection by electrospray mass spectrometry (ESI-MS). These additives were first tested for the detection of standard proteins by ESI-MS by flow-injection analysis (FIA). Those additives yielding the highest signals were employed for the separation of standard proteins by using three different reversed-phase columns: two C18 columns (4.6 mm I.D. and 2.1 mm I.D.) and one perfusion column (2 mm I.D.). The sensitivity of the LC-MS system was evaluated with the column giving the best results and with those LC eluents enabling the LC separation of the proteins and also yielding the highest MS signals. For that purpose, calibration curves were compared for both LC-MS and FIA-MS. Formic acid was the additive yielding the highest responses in FIA-MS and trifluoroacetic acid (TFA) gave the best separation and recovery of the proteins. However, problems related to poor recovery of the proteins in the column when formic acid was used and the significant signal suppression observed in MS when TFA was employed, made neither of them suitable for the sensitive detection of the proteins in LC-MS.

Screening of natural products extracts for the presence of phosphodiesterase inhibitors using liquid chromatography coupled online to parallel biochemical detection and chemical characterization

The ability to rapidly identify active compounds in a complex mixture (e.g., natural products extract) is still one of the major problems in natural products screening programs. An elegant way to overcome this problem is to separate the complex mixture by gradient liquid chromatography followed by online biochemical detection parallel with chemical characterization, referred to as high-resolution screening (HRS). To find and identify phosphodiesterase (PDE) inhibitors in natural products extracts using the HRS technology, the authors developed a continuous-flow PDE enzymatic assay. The suitability of the continuous-flow PDE enzymatic assay for natural products screening was demonstrated. After optimization of the continuous-flow PDE assay, the limit of detection for 3-isobutyl-1-methyl-xanthine (IBMX) was 1 [.proportional]M, with a dynamic range from 1 to 100 [.proportional]M IBMX. The applicability of the HRS technology for the detection of PDE inhibitors in natural products extracts was demonstrated by the analysis of a plant extract spiked with 2 naturally occurring PDE inhibitors. The plant extract was analyzed with 2 assay lines in parallel, enabling background fluorescence correction of the sample. The simultaneous quantification of the active compounds using evaporative light-scattering detection allowed the estimation of the IC50 value of the active compounds directly in the crude extract.

Online magnetic bead dynamic protein-affinity selection coupled to LC-MS for the screening of pharmacologically active compounds

The online, selective isolation of protein-ligand complexes using cobalt(II)-coated paramagnetic affinity beads (PABs) and subsequent liquid chromatography-mass spectrometry (LC-MS) determination of specifically bound ligands is described. After in-solution incubation of an analyte mixture with His-tagged target proteins, protein-analyte complexes are mixed with the Co(II)-PABs and subsequently injected into an in-house built magnetic trapping device. Bioactive ligands bound to the protein-Co(II)-PABs are retained in the magnetic field of the trapping device while inactive compounds are removed by washing with a pH 7.4 buffer. Active ligands are online eluted toward the LC-MS system using a pH shift. In the final step of the procedure, the protein-Co(II)-PABs are flushed to waste by temporarily lowering the magnetic field. The proof-of-principle is demonstrated by using commercially available Co(II)-PABs in combination with the His-tagged human estrogen-receptor ligand-binding domain. The system is characterized with a number of estrogenic ligands and nonbinding pharmaceutical compounds. The affinities of the test compounds varied from the high micromolar to the subnanomolar range. Typical detection limits are in the range from 20 to 80 nmol/L. The system is able to identify binders in mixtures of compounds, with an analysis time of 9.5 min per mixture. The standard deviation over 24 h is 9%.

Advances in mass spectrometry-based post-column bioaffinity profiling of mixtures

AbstractIn the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated bioaffinity screening. Four major strategies can be used for screening of bioactive mixtures for protein targets—pre-column and post-column off-line, at-line, and on-line strategies. The focus of this review is on recent developments in post-column on-line screening, and the role of mass spectrometry (MS) in these systems. On-line screening systems integrate separation sciences, mass spectrometry, and biochemical methodology, enabling screening for active compounds in complex mixtures. There are three main variants of on-line MS based bioassays: the mass spectrometer is used for ligand identification only; the mass spectrometer is used for both ligand identification and bioassay readout; or MS detection is conducted in parallel with at-line microfractionation with off-line bioaffinity analysis. On the basis of the different fields of application of on-line screening, the principles are explained and their usefulness in the different fields of drug research is critically evaluated. Furthermore, off-line screening is discussed briefly with the on-line and at-line approaches. Schematic view of an on-line bioaffinity analysis or HRS setup with MS based bioassay detection

Novel monitoring strategies for xenoestrogens

Abstract The wide variety of known and unknown xenoestrogenic compounds requires intelligent solutions to detect polluted environmental samples and identify the structure and concentration of the relevant xenoestrogens. In this article, a summary is given of several methods that are at present employed in the monitoring of xenoestrogens. Screening methods based on biological assays for the class-selective detection of xenoestrogens are reviewed. Furthermore, the analysis of known (xeno-)estrogens is summarized. Novel interfaces between biological assays and chromatography that facilitate the identification and quantification of xenoestrogens in complex samples are also discussed.

Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays, high resolution mass spectrometry and off-line NMR.

This paper describes the determination and identification of active and inactive estrogenic compounds produced by biosynthetic methods. A hyphenated screening assay towards the human estrogen receptor ligand binding domain (hER)alpha and hERbeta integrating target-ligand interactions and liquid chromatography-high resolution mass spectrometry was used. With this approach, information on both biologic activity and structure identity of compounds produced by bacterial mutants of cytochrome P450s was obtained in parallel. Initial structure identification was achieved by high resolution MS/MS, while for full structure determination, P450 incubations were scaled up and the produced entities were purified using preparative liquid chromatography with automated fraction collection. NMR spectroscopy was performed on all fractions for 3D structure analysis; this included 1D-(1)H, 2D-COSY, 2D-NOESY, and (1)H-(13)C-HSQC experiments. This multidimensional screening approach enabled the detection of low abundant biotransformation products which were not suitable for detection in either one of its single components. In total, the analytical scale biosynthesis produced over 85 compounds from 6 different starting templates. Inter- and intra-day variation of the biochemical signals in the dual receptor affinity detection system was less than 5%. The multi-target screening approach combined with full structure characterization based on high resolution MS(/MS) and NMR spectroscopy demonstrated in this paper can generally be applied to e.g. metabolism studies and compound-library screening.