Mass spectrometric real-time monitoring of an enzymatic phosphorylation assay using internal standards and data-handling freeware.

RATIONALE: Continuous-flow reaction detection systems (monitoring enzymatic reactions with mass spectrometry (MS)) lack quantitative values so far. Therefore, two independent internal standards (IS) are implemented in a way that the online system stability can be observed, quantitative conversion values for substrate and product can be obtained and they can be used as mass calibration standards for high MS accuracy. METHODS: An application previously developed for the MS detection of peptide phosphorylation by cAMP-dependent protein kinase A (PKA) (De Boer et al., Anal. Bioanal. Chem. 2005, 381, 647-655) was transferred to a continuous-flow reaction detection system. This enzymatic reaction, involving enzyme activation as well as the transfer of a phosphate group from ATP to a peptide substrate, was used to prove the compatibility of a quantitative enzymatic assay in a continuous-flow real-time system (connected to MS). RESULTS: Moreover (using internal standards), the critical parameter reaction temperature (including solution density variations depending on temperature) was studied in the continuous-flow mixing system. Furthermore, two substrates (malantide and kemptide), two enzyme types (catalytic subunit of PKA and complete PKA) and one inhibitor were tested to determine system robustness and long-term availability. Even spraying solutions that contained significant amount of MS contaminants (e.g. the polluted catalytic subunit) resulted in quantifiable MS signal intensities. Subsequent recalculations using the internal standards led to results representing the power of this application. CONCLUSIONS: The presented methodology and the data evaluation with available Achroma freeware enable the direct coupling of biochemical assays with quantitative MS detection. Monitoring changes such as temperature, reaction time, inhibition, or compound concentrations can be observed quantitatively and thus enzymatic activity can be calculated.

Adolescent nicotine exposure transiently increases high-affinity nicotinic receptors and modulates inhibitory synaptic transmission in rat medial prefrontal cortex.

Adolescence is a critical developmental period during which most adult smokers initiate their habit. Adolescents are more vulnerable than adults to nicotine's long-term effects on addictive and cognitive behavior. We investigated whether adolescent nicotine exposure in rats modifies expression of nicotinic acetylcholine receptors (nAChRs) in medial prefrontal cortex (mPFC) in the short and/or long term, and whether this has functional consequences. Using receptor binding studies followed by immunoprecipitation of nAChR subunits, we showed that adolescent nicotine exposure, as compared with saline, caused an increase in mPFC nAChRs containing α4 or ß2 subunits (24 and 18%, respectively) 24 h after the last injection. Nicotine exposure in adulthood had no such effect. This increase was transient and was not observed 5 wk following either adolescent or adult nicotine exposure. In line with increased nAChRs expression 1 d after adolescent nicotine exposure, we observed a 34% increase in amplitude of nicotine-induced spontaneous inhibitory postsynaptic currents in layer II/III mPFC pyramidal neurons. These effects were transient and specific, and observed only acutely after adolescent nicotine exposure, but not after 5 wk, and no changes were observed in adult-exposed animals. The acute nicotine-induced increase in α4ß2-containing receptors in adolescents interferes with the normal developmental decrease (37%) of these receptors from early adolescence (postnatal day 34) to adulthood (postnatal day 104) in the mPFC. Together, this suggests that these receptors play a role in mediating the acute rewarding effects of nicotine and may underlie the increased sensitivity of adolescents to nicotine.

On-line electrochemistry-bioaffinity screening with parallel HR-LC-MS for the generation and characterization of modified p38α kinase inhibitors.

In this study, an integrated approach is developed for the formation, identification and biological characterization of electrochemical conversion products of p38α mitogen-activated protein kinase inhibitors. This work demonstrates the hyphenation of an electrochemical reaction cell with a continuous-flow bioaffinity assay and parallel LC-HR-MS. Competition of the formed products with a tracer (SKF-86002) that shows fluorescence enhancement in the orthosteric binding site of the p38α kinase is the readout for bioaffinity. Parallel HR-MS(n) experiments provided information on the identity of binders and non-binders. Finally, the data produced with this on-line system were compared to electrochemical conversion products generated off-line. The electrochemical conversion of 1-{6-chloro-5-[(2R,5S)-4-(4-fluorobenzyl)-2,5-dimethylpiperazine-1-carbonyl]-3aH-indol-3-yl}-2-morpholinoethane-1,2-dione resulted in eight products, three of which showed bioaffinity in the continuous-flow p38α bioaffinity assay used. Electrochemical conversion of BIRB796 resulted, amongst others, in the formation of the reactive quinoneimine structure and its corresponding hydroquinone. Both products were detected in the p38α bioaffinity assay, which indicates binding to the p38α kinase.

Nanofractionation spotter technology for rapid contactless and high-resolution deposition of LC eluent for further off-line analysis.

The development of a contactless postcolumn spotter technology capable of rapidly and accurately depositing LC eluent onto another platform (e.g., 1536-well microtiter plates) is described. Many detection methodologies are suitable for online analysis, such as mass spectrometry, UV-vis, and fluorescence. In some cases, when online analysis is less suitable, off-line postcolumn analysis is the methodology of choice and usually relies on LC-based fractionation prior to detection (e.g., MALDI-MS, Raman spectrsocopy, biochemical assays). As fractionation generally involves loss in resolution, the technology described here allows high-resolution contactless fractionation by tailoring the fractionation frequency to the chromatographic peaks and mixing in of postcolumn reagents. Droplet ejection at frequencies of at least 6 Hz could be performed in the nanoliter to low microliter range with repeatabilities of ∼6%. Furthermore, multiple droplets can be ejected at the same position thereby allowing adjustment of fractionation volume and speed. The technology was evaluated, optimized, and validated prior to two proof-of-principle demonstrations comprising off-line chemical detection of injected fluorescein and off-line postcolumn biochemical detection of acetylcholine-binding protein ligands, both based on 1536-well plate reader analysis.

Studying protein-protein affinity and immobilized ligand-protein affinity interactions using MS-based methods.

This review discusses the most important current methods employing mass spectrometry (MS) analysis for the study of protein affinity interactions. The methods are discussed in depth with particular reference to MS-based approaches for analyzing protein-protein and protein-immobilized ligand interactions, analyzed either directly or indirectly. First, we introduce MS methods for the study of intact protein complexes in the gas phase. Next, pull-down methods for affinity-based analysis of protein-protein and protein-immobilized ligand interactions are discussed. Presently, this field of research is often called interactomics or interaction proteomics. A slightly different approach that will be discussed, chemical proteomics, allows one to analyze selectivity profiles of ligands for multiple drug targets and off-targets. Additionally, of particular interest is the use of surface plasmon resonance technologies coupled with MS for the study of protein interactions. The review addresses the principle of each of the methods with a focus on recent developments and the applicability to lead compound generation in drug discovery as well as the elucidation of protein interactions involved in cellular processes. The review focuses on the analysis of bioaffinity interactions of proteins with other proteins and with ligands, where the proteins are considered as the bioactives analyzed by MS.

Recent developments in protein-ligand affinity mass spectrometry.

This 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.

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

In 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.

Development of surface plasmon resonance biosensor assays for primary and secondary screening of acetylcholine binding protein ligands.

Surface plasmon resonance (SPR) biosensors recently gained an important place in drug discovery. Here we present a primary and secondary SPR biosensor screening methodology. The primary screening method is based on a direct binding assay with covalent immobilized drug target proteins. For the secondary screening method, a sequential competition assay has been developed where the captured protein is first exposed to an unknown test compound, followed directly by an exposure to a high-molecular-weight reporter ligand. Using the high-molecular-weight reporter ligand to probe the remaining free binding site on the sensor, a significant signal enhancement is obtained. Furthermore, this assay format allows the validation of the primary direct binding assay format, efficiently revealing false positive data. As a model system, acetylcholine binding protein (AChBP), which is a soluble model protein for neuronal nicotinic acetylcholine receptors, has been used. The secondary assay is lower in throughput than the primary assay; however, the signal-to-noise ratio is two times higher compared with the direct assay, and it has a z' factor of 0.96. Using both assays, we identified the compound tacrine as a ligand for AChBP.

Structural elucidation of biologically active neomycin N-octyl derivatives in a regioisomeric mixture by means of liquid chromatography/ion trap time-of-flight mass spectrometry.

Structural elucidation of six regioisomers of mono-N-octyl derivatized neomycin is achieved using MS(n) (up to n = 4) on an ion trap time-of-flight (IT-TOF) instrument equipped with electrospray ionization. The mixture of six derivatized neomycin analogues was generated by reductive amination in a shotgun synthetic approach. In parallel to the liquid chromatography/mass spectrometry (LC/MS) detection, the antibacterial activity of the neomycin regioisomers was tested by post-column addition of buffer and bacterial inocula, subsequent microfractionation of the resulting mixture, incubation, and finally a chemiluminescence-based bioactivity measurement based on the production of bacterial ATP. The MS-based high-resolution screening approach described can be applied in medicinal chemistry to help in designing and producing new antibiotic substances, which is particularly challenging due to the high functionality of most antibiotic substances, therefore requiring advanced (hyphenated) separation and detection techniques for compound mixtures.

Development of an online p38α mitogen-activated protein kinase binding assay and integration of LC-HR-MS.

A high-resolution screening method was developed for the p38α mitogen-activated protein kinase to detect and identify small-molecule binders. Its central role in inflammatory diseases makes this enzyme a very important drug target. The setup integrates separation by high-performance liquid chromatography with two parallel detection techniques. High-resolution mass spectrometry gives structural information to identify small molecules while an online enzyme binding detection method provides data on p38α binding. The separation step allows the individual assessment of compounds in a mixture and links affinity and structure information via the retention time. Enzyme binding detection was achieved with a competitive binding assay based on fluorescence enhancement which has a simple principle, is inexpensive, and is easy to interpret. The concentrations of p38α and the fluorescence tracer SK&F86002 were optimized as well as incubation temperature, formic acid content of the LC eluents, and the material of the incubation tubing. The latter notably improved the screening of highly lipophilic compounds. For optimization and validation purposes, the known kinase inhibitors BIRB796, TAK715, and MAPKI1 were used among others. The result is a high-quality assay with Z' factors around 0.8, which is suitable for semi-quantitative affinity measurements and applicable to various binding modes. Furthermore, the integrated approach gives affinity data on individual compounds instead of averaged ones for mixtures.

Targeted LC-MS derivatization for aldehydes and carboxylic acids with a new derivatization agent 4-APEBA.

Based on the template of a recently introduced derivatization reagent for aldehydes, 4-(2-(trimethylammonio)ethoxy)benzeneaminium dibromide (4-APC), a new derivatization agent was designed with additional features for the analysis and screening of biomarkers of lipid peroxidation. The new derivatization reagent, 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APEBA) contains a bromophenethyl group to incorporate an isotopic signature to the derivatives and to add additional fragmentation identifiers, collectively enhancing the abilities for detection and screening of unknown aldehydes. Derivatization can be achieved under mild conditions (pH 5.7, 10 degrees C). By changing the secondary reagent (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide instead of sodium cyanoborohydride), 4-APEBA is also applicable to the selective derivatization of carboxylic acids. Synthesis of the new label, exploration of the derivatization conditions, characterization of the fragmentation of the aldehyde and carboxylic acid derivatives in MS/MS, and preliminary applications of the labeling strategy for the analysis of aldehydes in urine and plasma are described.

Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on-line biochemical assays.

One way to profile complex mixtures for receptor affinity is to couple liquid chromatography (LC) on-line to biochemical detection (BCD). A drawback of this hyphenated screening approach is the relatively high consumption of sample, receptor protein and (fluorescently labeled) tracer ligand. Here, we worked toward minimization of sample and reagent consumption, by coupling nano-LC on-line to a light-emitting diode (LED) based capillary confocal fluorescence detection system capable of on-line BCD with low-flow rates. In this fluorescence detection system, a capillary with an extended light path (bubble cell) was used as a detection cell in order to enhance sensitivity. The technology was applied to a fluorescent enhancement bioassay for the acetylcholine binding protein, a structural analog of the extracellular ligand-binding domain of neuronal nicotinic acetylcholine receptors. In the miniaturized setup, the sensitive and low void volume LED-induced confocal fluorescence detection system operated in flow injection analysis mode allowing the measurement of IC(50) values, which were comparable with those measured by a conventional plate reader bioassay. The current setup uses 50 nL as injection volume with a carrier flow rate of 400 nL/min. Finally, coupling of the detection system to gradient reversed-phase nano-LC allowed analysis of mixtures in order to identify the bioactive compounds present by injecting 10 nL of each mixture.

Direct Dynamic Protein-Affinity Selection Mass-Spectrometry.

A new methodology is described enabling the affinity screening of potential ligands towards the human estrogen receptor alpha ligand binding domain (ERalpha-LBD). In-solution incubation is performed of the analyte and the His-tagged ERalpha-LBD. The bound complex is immobilized on a nickel-loaded protein-affinity selection column, where after the unbound fraction is removed. The immobilized protein-ligand complex is exposed to a decreased pH value and an increased organic modifier concentration releasing the ligand for MS detection, and precipitating the proteins on a filter positioned between the affinity column and the mass spectrometer. The trapping column can be regenerated for reuse at least 70 times. The advantages of the methodology over existing methodologies are the absence of a pre-concentration as well as a chromatographic separation step, resulting in a significantly shorter analysis time compared to previously described procedures, and in addition, allowing the determination of solutes with unfavorable chromatographic properties. The overall analysis time now can be reduced about 250% to approximately 6 min. Replacing the filters after every measurement results in an intra-day standard deviation of 14.8% and an inter-day standard deviation of 21.3%.

Identification of the Biotransformation Products of 2-Ethylhexyl 4-(N,N-Dimethylamino)benzoate.

Nowadays, 2-ethylhexyl 4-(N,N-dimethylamino)benzoate (EDP) is one of the most widely used UV filters in sunscreen cosmetics and other cosmetic products. However, undesirable processes such as percutaneous absorption and biological activity have been attributed to this compound. The in vitro metabolism of EDP was elucidated in the present work. First of all, the phase I biotransformation was studied in rat liver microsomes and two metabolites, N,N-dimethyl-p-aminobenzoic acid (DMP) and N-monomethyl-p-aminobenzoic acid (MMP), were identified by GC-MS analysis. Secondly, the phase II metabolism was investigated by means of LC-MS. The investigated reactions were acetylation and glucuronidation working with rat liver cytosol and with both human and rat liver microsomes, respectively. Analogue studies with p-aminobenzoic acid (PABA) were carried out in order to compare the well established metabolic pathway of PABA with the unknown biotransformation of EDP. In addition, a method for the determination of EDP and its two phase I metabolites in human urine was developed. The methodology requires a solid-phase extraction prior to LC-MS analysis. The method is based on standard addition quantification and has been fully validated. The repeatability of the method, expressed as relative standard deviation, was in the range 3.4-7.4% and the limit of detection for all quantified analytes was in the low ng mL(-1) range.

Microfractionation revisited: a 1536 well high resolution screening assay.

The aim of the here presented study was to combine high performance liquid chromatography with plate reader technology in order to overcome certain drawbacks of integrated online systems as well as offline plate reader approaches. The described method combines an "at-line" enzyme assay for the simultaneous bioactivity determination with parallel QTOF MS data acquisition for analyte identification. All biochemical reagents are added in an online mode directly to the column effluent (postcolumn addition/mixing), and the complete screening assay mixture is subsequently microfractionated into a 1536 well plate. The screening of a natural extract fortified with two well-known Protein Kinase A inhibitors and the identification of an inhibitor in a natural extract showed the applicability of the approach to detect bioactive compounds in low concentrations in a complex mixture. The described mode of operation utilizes today's plate reader technology to its full capacity and directly hyphenates it to a high resolution separation technique which has not been shown before. Furthermore, it allows coupling of a microbore HPLC with a biochemical screening assay without compromising resolution and overcomes problems associated with the 1536 well format.

High-throughput reaction optimisation and activity screening of ferrocene-based Lewis acid-catalyst complexes by using continuous-flow reaction detection mass spectrometry.

Optimising synthetic conversions and assessing catalyst performance is a tedious and laborious endeavour. Herein, we present an automated alternative to the commonly applied sequential approaches that are used to increase catalyst discovery process efficiencies by increasing the number of entities that can be tested. This new approach combines conversion of the reactants and determination of product formation into a single comprehensive reaction detection system that can be operated with minimal catalyst and reactant consumption. With this approach, rudimentary reaction conditions can be quickly optimised and the same system can then be used to screen for the optimal homogenous catalyst in a selected solution-phase synthetic conversion. The system, which is composed of standard HPLC components, can be used to screen catalyst libraries at a repetition rate of five minutes and can be run unsupervised. The sensitive mass spectrometric detection that is implemented in the reaction detection methodology can be used for the simultaneous monitoring of reactants, catalysts and product ions. In the experiments, the three-component reaction that gives a substituted 2-imidazoline was optimised. Afterwards, the same method was used to assess a library of ferrocene-based Lewis acid catalysts for performance in the aforementioned conversion in six different solvents. We demonstrate the feasibility of using this methodology to directly compare the performance results obtained in different solvents by calibrating the solvent-specific MS responses.

Analysis of glutathione adducts of patulin by means of liquid chromatography (HPLC) with biochemical detection (BCD) and electrospray ionization tandem mass spectrometry (ESI-MS/MS).

A novel method for the identification of glutathione/electrophile adducts that are inhibiting glutathione-S-transferase (GST) activity was developed and applied for the analysis of the mycotoxin patulin. The method is based on high-performance liquid chromatography (HPLC) coupled to a continuous-flow enzyme reactor serving as biochemical detector (BCD) in parallel to electrospray mass spectrometric detection (ESI-MS). This HPLC-BCD technique combines a separation step and the detection of the inhibition and is therefore ideally suited for the analysis of the activity of single patulin/glutathione adducts within a complex mixture of adducts. Two out of at least 15 detected patulin-glutathione adducts showed strong GST inhibition. In ESI-MS, the inhibitory active adducts were characterized by [M + H]+ ions with m/z 462.1138 and m/z 741.2011, respectively. They could be identified as a dihydropyranone adduct containing one molecule glutathione and a ketohexanoic acid bearing two glutathione molecules.

Development of a countergradient parking system for gradient liquid chromatography with online biochemical detection of serine protease inhibitors.

A gradient HPLC approach in combination with a countergradient system for online biochemical detection (BCD) to screen for inhibitors of serine proteases is described. For gradient separations, this novel countergradient system was developed to produce a biocompatible constant solvent composition in the BCD. The countergradient system is based on retaining complete gradients in an additional preparative HPLC column, followed by subsequent and reversible elution to the separation column effluent. Major advantages compared with existing countergradient systems are that no additional LC pumps are needed and enhanced stability. The developed countergradient system was systematically characterized applying different gradient programs. Inhibitors eluting in a postcolumn continuous flow analysis interfere with the enzymatic release of fluorescent 7-amino-4-methylcoumarin (AMC) from an AMC-labeled peptide. The inhibitory activity of eluting substances is sensitively detected as the degree of reduced fluorescence intensity. This biochemical detection system (BCD) for proteases was validated with three known inhibitors of the benzamidine type. Their IC 50 values were in good accordance with the results of conventional plate reader assays. Finally, a small library of protease inhibitors was successfully screened with the combination of the BCD and the countergradient system.

Online screening of homogeneous catalyst performance using reaction detection mass spectrometry.

An integrated online screening system was developed to rapidly screen homogeneous catalysts for activity toward a selected synthesis. The continuous-flow system comprises standard HPLC pumps for the delivery of substrates, an HPLC autosampler for the injection of homogeneous catalysts, a thermostated reactor to mediate synthesis, and a single-stage quadrupole mass spectrometer (MS) equipped with atmospheric pressure chemical ionization for the determination of product formation. MS detection offers sensitivity, specificity, and speed when applied to the analysis of dynamic processes in the condensed phase. By applying the present methodology for the study of substrate conversion mediated by homogeneous catalysts, the concentration of substrates and reaction product could be monitored while information about the catalysts could also be obtained. In an initial screening application, the performance of a selected number of Lewis acids in the multicomponent synthesis of a highly substituted 2-imidazoline was determined. Limit of detection and limit of quantitation were determined by injecting different concentrations of 2-imidazoline standards and proved to be 1.6 and 5.2 nM, respectively. The results obtained with the new screening method were in good agreement with a traditional bench-scale experiment. Moreover, the system was capable of determining catalyst performance with very low catalyst and solvent consumption while the ruggedness of the system was exhibited with a 24-h continuous analysis of 280 successive catalyst injections with a peak area variation within 7% relative standard deviation.

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.