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.

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.

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.

High-resolution bioactivity profiling of mixtures toward the acetylcholine binding protein using a nanofractionation spotter technology.

This study describes the evaluation, validation, and use of contactless postcolumn fractionation of bioactive mixtures with acetylcholine binding protein (AChBP) affinity analysis with help of a spotter technology. The high-resolution fractionation tailors the fractionation frequency to the chromatographic peaks. Postcolumn reagents for AChBP bioaffinity profiling are mixed prior to droplet ejection into 1536-well plates.

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.

High temperature liquid chromatography hyphenated with ESI-MS and ICP-MS detection for the structural characterization and quantification of halogen containing drug metabolites.

In this paper we describe the hyphenation of high temperature liquid chromatography with ICP-MS and ESI-MS for the characterization of halogen containing drug metabolites. The use of temperature gradients up to 200°C enabled the separation of metabolites with low organic modifier content. This specific property allowed the use of detection methods that suffer from (significant) changes in analyte response factors as a function of the organic modifier content such as ICP-MS.

Optimization of information content in a mass spectrometry based flow-chemistry system by investigating different ionization approaches.

Current development in catalyst discovery includes combinatorial synthesis methods for the rapid generation of compound libraries combined with high-throughput performance-screening methods to determine the associated activities. Of these novel methodologies, mass spectrometry (MS) based flow chemistry methods are especially attractive due to the ability to combine sensitive detection of the formed reaction product with identification of introduced catalyst complexes. Recently, such a mass spectrometry based continuous-flow reaction detection system was utilized to screen silver-adducted ferrocenyl bidentate catalyst complexes for activity in a multicomponent synthesis of a substituted 2-imidazoline.

Stability-indicating study of the anti-Alzheimer's drug galantamine hydrobromide.

Galantamine hydrobromide was subjected to different stress conditions (acidic, alkaline, thermal, photolytic and oxidative). Degradation was found to occur under acidic, photolytic and oxidative conditions, while the drug was stable under alkaline and elevated temperature conditions. A stability-indicating reversed-phase liquid chromatographic method was developed for the determination of the drug in the presence of its degradation products.

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.

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.

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.

Tandem mass spectrometry of silver-adducted ferrocenyl catalyst complexes.

Ferrocene is a popular template in material science due to its exceptional characteristics that offer the ability to optimize the selectivity and activity of catalysts by the addition of carefully selected substituents. In combinatorial catalyst development, the high susceptibility to electrophilic substitution reactions offers the opportunity for the rapid introduction of molecular diversity. Mass spectrometry (MS)-based continuous-flow systems can be applied to rapidly evaluate catalyst performance as well as to (provisionally) identify the introduced catalyst complexes.

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.

Surface plasmon resonance biosensor based fragment screening using acetylcholine binding protein identifies ligand efficiency hot spots (LE hot spots) by deconstruction of nicotinic acetylcholine receptor α7 ligands.

The soluble acetylcholine binding protein (AChBP) is a homologue of the ligand-binding domain of the nicotinic acetylcholine receptors (nAChR). To guide future fragment-screening using surface plasmon resonance (SPR) biosensor technology as a label-free, direct binding, biophysical screening assay, a focused fragment library was generated based on deconstruction of a set of α7 nAChR selective quinuclidine containing ligands with nanomolar affinities. The interaction characteristics of the fragments and the parent compounds with AChBP were evaluated using an SPR biosensor assay.

Target immobilization as a strategy for NMR-based fragment screening: comparison of TINS, STD, and SPR for fragment hit identification.

Fragment-based drug discovery (FBDD) has become a widely accepted tool that is complementary to high-throughput screening (HTS) in developing small-molecule inhibitors of pharmaceutical targets. Because a fragment campaign can only be as successful as the hit matter found, it is critical that the first stage of the process be optimized. Here the authors compare the 3 most commonly used methods for hit discovery in FBDD: high concentration screening (HCS), solution ligand-observed nuclear magnetic resonance (NMR), and surface plasmon resonance (SPR).

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.

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.

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.

Online fluorescence enhancement assay for the acetylcholine binding protein with parallel mass spectrometric identification.

The acetylcholine binding protein (AChBP) is considered an analogue for the ligand-binding domain of neuronal nicotinic acetylcholine receptors (nAChRs). Its stability and solubility in aqueous buffer allowed the development of an online bioaffinity analysis system. For this, a tracer ligand which displays enhanced fluorescence in the binding pocket of AChBP was identified from a concise series of synthetic benzylidene anabaseines.

Production and on-line acetylcholinesterase bioactivity profiling of chemical and biological degradation products of tacrine.

The present paper describes a methodology for rapid assessment of chemical and biological degradation products of tacrine and their bioactivity for acetylcholinesterase (AChE). Analysis was achieved by utilizing liquid chromatography coupled to parallel high resolution mass spectrometry and an on-line continuous-flow AChE bioassay for biochemical detection. Key advantage of the strategy described involves the straightforward chemical production of large quantities of products of which many were the same as formed during the biological degradation by cytochromes P450 (CYPs).

High throughput screening methodologies classified for major drug target classes according to target signaling pathways.

Over the years, many different high throughput screening technologies and subsequently follow-up methodologies have been developed. All of these can be categorized, for example according to measurement of analyte classes, assay mechanisms, readout principles, or screening of drug target classes. When categorized according to drug target class, assay formats can be subdivided into early hit stage assays (usually ligand-binding based) that are often straightforward and robust up to analysis of final cellular effects exerted by ligands.

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.

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.

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.

Photohuperzine A-A new photoisomer of huperzine A: structure elucidation, formation kinetics and activity assessment.

A new photoisomer of the promising "anti-Alzheimer" drug candidate (+/-) huperzine A is described. The new substance was formed via a photoisomerization reaction and was found to be 1-amino-13-ethylidene-11-methyl-6-aza-tetracyclo-[7.3.