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%.

Metal-complex formation in continuous-flow ligand-exchange reactors studied by electrospray mass spectrometry.

Electrospray ionization mass spectrometry was used to investigate complex formation of different metal complexes in a continuous-flow ligand-exchange reactor. A computer program was developed based on normal equilibrium calculations to predict the formation of various metal-ligand complexes. Corresponding to these calculations, infusion electrospray mass spectrometric experiments were performed to investigate the actual complex formation in solution. The data clearly show good correlation between the theoretically calculated formation of metal-ligand complexes and the experimental mass spectrometric data. Moreover, the approach demonstrates that the influence of the pH can be investigated using a similar approach. Indirectly, these infusion experiments provide information on relative binding constants of different ligands towards a metal-ion. To demonstrate this, a continuous-flow ligand-exchange detection system with mass spectrometric detection was developed. Injection of ligands, with different affinity for the metal-ion, into the reactor shows good correlation between binding constants and the response in the ligand-exchange detection system. Additional information on the introduced ligand, and the complexes formed after introduction of the ligand, can be obtained from interpretation of the mass spectra.

Spreeta-based biosensor assays for endocrine disruptors.

The construction and performance of an automated low-cost Spreeta-based prototype biosensor system for the detection of endocrine disrupting chemicals (EDCs) is described. The system consists primarily of a Spreeta miniature liquid sensor incorporated into an aluminum flow cell holder, dedicated to support a Biacore chip frame, in combination with a simple pressurized air-driven fluid system. During the optimization, a monoclonal antibody (MAb)-based immunoassay for the estrogenic compound bisphenol A (BPA) was used as a model. After the optimization two thyroxine transport protein inhibition assays for thyroid endocrine disruptors were implemented. The average noise of the system for 1 min of baseline was 1.1 microRIU (refractive index units) and it could be operated in the range of 18-22 degrees C with a minimum baseline drift (5-10 microRIU/100 min). Optimum signal to noise ratio (S/N R) was obtained using a flow cell height of 100 microm and a flow rate of 180 microl/min. The sensitivity of the Spreeta-based biosensor inhibition assays implemented (50% inhibition concentration (IC50) of 30.2 nM for BPA using MAb12 and 12.3 and 11.6 nM for thyroxine (T4) using thyroxine-binding globulin (TBG) and recombinant transthyretin (rTTR), respectively) was comparable to the sensitivity previously obtained using a Biacore 3000 (IC50 of 39.9 nM for BPA and 8.6 and 13.7 nM, respectively, for T4). The results indicate that the alternative prototype system can be used in combination with ready-to-use biosensor chip surfaces and it is potentially a useful tool for the bioeffect-related screening of EDCs.

Selective detection and identification of phosphorylated proteins by simultaneous ligand-exchange fluorescence detection and mass spectrometry.

A ligand-exchange method for the detection and identification of phosphorylated peptides in complex mixtures is presented that is based on the characterization of phosphorylated species by solution-phase interactions with Fe(III) ions and subsequent fluorescence readout. After the separation of the peptides and digest products on a reversed-phase LC column, the flow is split between the two detection systems. One part is directed towards an electrospray mass spectrometer for direct detection and identification of all the peptides present in the sample. The other part of the flow is directed towards a ligand-exchange detection system. This system relies on the specific release of a fluorescent reporter ligand from a Fe(III)-complex in the presence of phosphorylated peptides. To recognize false positive signals due to high-affinity non-phosphorylated high-acidic peptides and other compounds which are known to be a problem in for instance immobilized metal affinity chromatography (IMAC), a second run is performed after incubation of the sample with alkaline phosphatase. A positive signal in this second run indicates a high-affinity non-phosphorylated compound. The method is illustrated using digest from a phosphorylated alpha-casein. Automated switching between MS and MS-MS was performed to obtain additional information about the compounds present in the sample. The linearity of the method was tested in the range of 0.5-80 microM of phosphorylated peptides. A limit of detection (LOD) of 0.5 microM was obtained for a mono-phosphorylated peptide. The interday (n=4) and intraday precision (n=3) expressed as relative standard deviation was better than 10%.

Screening for metal ligands by liquid chromatography--ligand-exchange--electrospray mass spectrometry.

Electrospray ionization mass spectrometry is applied for the selective detection of metal ligands after a post-column continuous-flow ligand-exchange reaction. The detection is based on the specific release of a reporter ligand from a metal-reporter ligand complex by a high affinity ligand. Constant infusion and direct-injection experiments are performed to optimize the method. The on-line coupling of a liquid chromatographic separation prior to the continuous flow ligand-exchange reaction enables the screening for high affinity ligands in complex samples. The feasibility of the method is demonstrated by using several ligands with a different affinity for either Cu(II) or Zn(II) ions. The selectivity of the ligand-exchange detection method can be tuned by the choice of the reporter ligand. This is demonstrated by using either 2,2'-bipyridyl or 5-methyl-1,10-phenanthroline as reporter ligands.

Label-free biochemical detection coupled on-line to liquid chromatography

The on-line coupling of a label-free optical biosensor to a HPLC system is described by combining the separation power of HPLC with the specificity of the biosensor system. A highly cross-reactive antibody against the pesticide isoproturon was used as model for affinity proteins. The binding strength of the antibody to the utilized pesticides was characterized with the biosensor, first. In the on-line coupling setup, the eluate of the HPLC was mixed continuously with the antibodies. The presence of antigens was detected by a reduction of the antibody binding to the transducer. This reduced binding was quantified by a differentiation of the sensor signal by applying a Savitzky-Golay algorithm. Limits of detection were found to be in the femtomole range without preconcentration, which is comparable to a study using fluorescence-based biochemical detection.

Optimisation of a heterogeneous non-competitive flow immunoassay comparing fluorescein, peroxidase and alkaline phosphatase as labels.

Off- and on-line strategies for a non-competitive heterogeneous flow immunoassay were developed comparing three different labels. The samples, containing the model compounds digoxin or digoxigenin, were either pre-incubated off-line or on-line in a mixing coil with excess of labelled anti-digoxigenin Fab-fragments. The excess of Fab-fragments was then separated from the digoxin bound Fab-fragments by passing the sample through a column with immobilised digoxin. The off-line immunochemical detection system is suitable for sensitive high through-put screening of the analytes, whereas the on-line system is more suitable for coupling as a post-column detection unit to liquid chromatography. The digoxin and digoxigenin content in the sample were quantified using fluorescein (F) and enzyme (peroxidase (POD), alkaline phosphatase (AP)) labelled Fab-fragments. The fluorescein label was directly measured with the fluorescence detector, whereas a fluorescent enzyme product was measured in the two enzyme based systems, using 3-(p-hydroxyphenyl)-propionic acid (HPPA) and hydrogen peroxide for POD and, and 4-methylumbelliferyl phosphate (4-MUP) for AP. The highest sensitivity and lowest limit of detection (LOD) was obtained with the Fab-POD system with LODs for digoxin and digoxigenin in the off- and on-line configurations of 0.025 and 0.01 nM, respectively. The sample through-put for the off- and on-line systems were 43 and 32 samples per hour, respectively.

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 muM, with a dynamic range from 1 to 100 muM 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 IC(50) value of the active compounds directly in the crude extract.

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) alpha and beta 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 flowthrough 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.

Determination of 1,2,6-inositol trisphosphate (derivatives) in plasma using iron(III)-loaded adsorbents and capillary zone electrophoresis-(indirect) UV detection.

A method for the determination of 1,2,6-inositol trisphosphate (IP3) and derivatives in plasma by capillary zone electrophoresis with (indirect) UV detection has been developed. The sample pretreatment is based on the selective isolation after complexation of inositol phosphates with iron(III) loaded on an adsorbent. Plasma protein denaturation was performed with sodium dodecyl sulfate. The selectivity of the method is demonstrated with the analysis of phenylacetate-IP3. The recoveries amount to 65% and 88% in plasma and in water, respectively.

Applying hollow fibres for separating free and bound label in continuous-flow immunochemical detection.

On-line liquid chromatography-immunochemical detection (LC-ICD) provides the possibility to individually monitor cross-reactive compounds overcoming the need of tedious fraction collection. ICD is performed as a post-column reaction detection system and is based on a two-step immunoreaction. In the first step unlabelled antibodies are added to the LC effluent and allowed to react with antigens (analytes) eluting from the LC column. The amount of analytes bound to the antibodies is measured by adding, in a second step, labelled antigen to the reaction mixture. For quantitation, free and bound label need to be separated prior to detection. The present paper describes a hollow fibre module (HFM), which can be used for this purpose. Separation of free and bound label occurs on discrimination by size. Using biotin as a model compound, a detection limit of 30 nmol/l can be reached employing anti-biotin antibodies and a low-molecular-mass fluorescence label in the LC-ICD system. Additional to low-molecular-mass labels, the HFM allows the use of small enzyme labels. In this context, horseradish peroxidase-labelled biotin was used as a label in combination with antibodies in the immunochemical detection of biotin. This allows future implementation of commercially available enzyme immunoassay kits in continuous-flow immunochemical detection.

Theoretical concepts of on-line liquid chromatographic-biochemical detection systems. I. Detection systems based on labelled ligands.

A theoretical foundation for on-line coupling of liquid chromatography (LC) with fluororeceptor assays based on fluorescent ligands is presented. Using a recently developed LC-receptor affinity detection (RAD) system as a model, equations are derived which describe the detector response and the signal-to-noise ratio as a function of important biochemical and instrumental parameters. The effect of ligand and label affinity, and receptor concentrations on the detector performance were investigated. It was found that the response of the RAD system is correlated with the affinity of the injected compounds and that the relative affinity order of binding ligands is maintained in the RAD system.

Potential of receptor-ligand interactions for sample handling in liquid and gas chromatography.

The use of receptor proteins for biospecific sample handling in liquid and gas chromatography is described. As a model system the uterine estrogen receptor was chosen for the isolation of 17 beta-estradiol and its synthetic agonist diethylstilbestrol. Biochemical characteristics relevant for the use of receptors in sample handling such as the kinetics of receptor-ligand binding, reproducibility and capacity are examined by means of an estrogen radioreceptor assay. Different techniques for the non-covalent immobilization of the estrogen receptor were investigated. Both protamine-coated glass fibre filters and silica particles which bind the receptor via electrostatic interactions have been used for this purpose. The use of the estrogen receptor in the isolation of 17 beta-estradiol and diethylstilbestrol prior to GC-MS analysis is demonstrated and discussed.

Theoretical concepts of on-line liquid chromatographic-biochemical detection systems. II. Detection systems based on labelled affinity proteins.

A theoretical concept for on-line liquid chromatography-biochemical detection (LC-BCD) using labelled affinity proteins as reporter molecules is presented. The BCD system is based on the post-column addition of labelled affinity proteins such as fluorescein-labelled streptavidin to the LC effluent. After a short reaction time, free and analyte-bound label are separated during passage through a column packed with an immobilised-ligand support. The bound fraction passes the column unretained and is measured downstream by means of a conventional HPLC detector. The theoretical model presented here relates the detector response to the most important instrumental and biochemical parameters such as dispersion, reaction time, concentration and affinity of the affinity protein and the number of binding sites. The theoretical concept is validated using fluorescein-labelled streptavidin and biotin as model system.

High-performance liquid chromatography coupled to enzyme-amplified biochemical detection for the analysis of hemoglobin after pre-column biotinylation.

The determination of proteins with enzyme-amplified biochemical detection (EA-BCD) coupled on-line with high-performance liquid chromatography (HPLC) is demonstrated. The EA-BCD system was developed to detect biotin-containing compounds. Hemoglobin, which was used as a model compound, was biotinylated prior to sample introduction. Several biotinylation parameters, such as pH and removal of excess biotinylation reagent, were investigated. After biotinylation samples were introduced to HPLC followed by EA-BCD. To the HPLC effluent, alkaline phosphatase label streptavidin (S-AP) was added, which possesses high affinity to biotin and biotin-containing compounds. Excess S-AP was removed by means of an immobilized biotin column followed by substrate addition. The non-fluorescent substrate is converted to a highly fluorescent product by the enzyme label. A detection limit of 2 femtomol biotinylated Hb was achieved with good reproducibility and linearity. However, biotinylation at low analyte concentration suffers from low yield due to slow reaction kinetics. Finally, Hb was successfully extracted from urine with a recovery of 94%.

Enzyme amplification as detection tool in continuous-flow systems. I. Development of an enzyme-amplified biochemical detection system coupled on-line to flow-injection analysis.

The on-line coupling of flow-injection analysis (FIA) to an enzyme-amplified biochemical detection (EA-BCD) system is described. The aim of this study is the development of a detection system able to detect biotin-containing compounds at low concentration levels. The detection system is based on the interaction of biotin with enzyme-labeled affinity proteins. Biotin possesses a high affinity to both streptavidin and anti-biotin Fab fragments, which are both tested. Several biotin derivatives are available with different reactive probes, which can be used to label analytes of interest. Therefore, biotin acts as a universal probe for the enzyme-amplified biochemical detection. Alkaline phosphatase (AP) was used as enzyme label. Several parameters, such as substrate type and concentration, concentration of enzyme-labeled affinity protein, reaction time and reaction temperature were examined. Biotin aminocaproic acid was used as a model compound. In addition to biotin aminocaproic hydrazide, other biotinylation reagents were also examined. With fluorescence detection of the enzyme-generated product, a mass detection limit of 1 fmol was achieved.

Enzyme amplification as detection tool in continuous-flow systems. II. On-line coupling of liquid chromatography to enzyme-amplified biochemical detection after pre-column derivatization with biotin.

Enzyme-amplified biochemical detection (EA-BCD) was used as a post-column detection technique, coupled on-line with high-performance liquid chromatography (HPLC). The enzyme detection system was developed to detect biotin or biotin containing compounds. Biotinylation is widely used to label analytes of interest ranging from small molecules to proteins and DNA. Naphthalene aldehyde and anthracene aldehyde were used as model compounds. Both compounds were biotinylated off-line with biotin aminocaproic hydrazide (BACH). On-column biotinylation was performed by preconcentration of anthracene aldehyde on copper phthalocyanine. After biotinylation, samples were introduced to the HPLC system. Enzyme-labeled streptavidin, which possesses high affinity to biotin, was added post-column to the HPLC effluent. Excess of enzyme-labeled affinity protein was removed by means of an immobilized biotin column. After separation of free and bound fraction, substrate was added, which was converted to a fluorescent product by the enzyme label. Using alkaline phosphatase as an enzyme label, a mass detection limit after on-column preconcentration and biotinylation of 250 fmol was achieved.

Liquid chromatography-mass spectrometry with on-line solid-phase extraction by a restricted-access C18 precolumn for direct plasma and urine injection.

In this paper, the on-line coupling of solid-phase extraction, based on a restricted-access support with liquid chromatography-mass spectrometry (LC-MS), for the analysis of biological samples is described. The system was tested with cortisol and prednisolone for plasma analysis and arachidonic acid for urine analysis. A precolumn packed with a 25-micron C18 alkyl-diol support is used for direct plasma or urine injection. Using column-switching techniques, the analytes enriched on the precolumn are eluted to the analytical column without transfer loss. An on-line heart-cut technique was employed and only the analyte-containing fraction eluting from the LC column is directed to the MS to protect the LC-MS interface and ion-source from contamination. The whole system is operated in a parallel mode, that is, sample pre-treatment and LC-MS analysis are performed simultaneously to provide the shortest possible analysis time. The only off-line sample pre-treatment step required was centrifugation to remove particulate matter. With the fully automated system, total analysis times of 5 and 9.5 min were achieved for cortisol in serum and arachidonic acid in urine, respectively. Cortisol and related compounds were quantitatively recovered from plasma with a detection limit for prednisolone (direct injection of 100 microliters on restricted-access precolumn) of 2 ng/ml.

High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products.

A high-performance liquid chromatography (HPLC) method with on-line coupled ultraviolet (UV), mass spectrometry (MS) and biochemical detection for acetylcholinesterase (AChE) inhibitory activity has been developed. By combining the separation power of HPLC, the high selectivity of biochemical detection, and the ability to provide molecular mass and structural information of MS, AChE inhibitors can be rapidly identified. The biochemical detection was based on a colorimetric method using Ellman's reagent. The detection limit of galanthamine, an AChE inhibitor, in the HPLC-biochemical detection is 0.3 nmol. The three detector lines used, i.e., UV, MS and Vis for the biochemical detection were recorded simultaneously and the delay times of the peaks obtained were found to be consistent. This on-line post-column detection technique can be used for the identification of AChE inhibitors in plant extracts and other complex mixtures such as combinatorial libraries.