Bio-solid-phase extraction/tandem mass spectrometry for identification of bioactive compounds in mixtures.

We describe a two-step column-based bioassay method with tandem mass spectrometric detection for rapid identification of bioactive species in mixtures. The first step uses an immobilized enzyme reactor (IMER) column interfaced to an electrospray ionization mass spectrometer (ESI-MS) to identify mixtures containing bioactive compounds (i.e., enzyme inhibitors), while the second step uses bioselective solid-phase extraction (bioSPE) columns to isolate compounds from "hit" mixtures, which are then identified online by data-dependent ESI-MS. IMER columns were prepared by entrapment of adenosine deaminase (ADA) into sol-gel derived monolithic silica columns, and used to perform a primary IMER screen of mixtures prepared from a bioactive library, which resulted in four apparent hit compounds. Such columns did not provide sufficient binding site density to allow bioSPE, and thus a new column format was developed using ADA that was covalently immobilized to monolithic silica capillary columns, providing ∼500-fold more protein binding sites than were present in columns containing entrapped proteins. Using the covalently linked ADA columns, bioactive mixtures identified by IMER were infused until a maximum total ion current was achieved, followed by washing with a buffer to remove unbound compounds. A harsh wash with 3% acetic acid eluted the strongly bound ligands and the resulting peak triggered data dependent MS/MS to identify the ligand, showing that two of the apparent hits were true ADA inhibitors and demonstrating the ability of this method to rapidly identify bioactive compounds in mixtures.

A sol-gel-derived acetylcholinesterase microarray for nanovolume small-molecule screening.

A fluorimetric acetylcholinesterase (AChE) assay was developed and characterized both in solution and with the enzyme entrapped in sol-gel-derived silica. The assay is based on a disulfide-thiol interchange reaction between the intramolecularly quenched dimeric dye BODIPY FL l-cystine and thiocholine generated by the AChE-catalyzed hydrolysis of acetylthiocholine (ATCh), which results in a brightly fluorescent monomeric product owing to the cleavage of the disulfide-coupled form of the dye. The new assay was validated by comparison with the Ellman assay performed under parallel conditions and was used in both kinetic and end point assays. The assay was extended to the fabrication of functional AChE microarrays using contact pin-printing of sol-gel-derived silica. A total of 392 sol-gel formulations were screened for gelation times and 192 of these were further evaluated for array fabrication on four different surfaces using a factor analysis approach. Of these, 66 sol-gel/surface combinations produced robust microarrays, while 26 sol-gel/surface combinations were identified that could produce highly active AChE microarrays. The Z' factor for the on-array assay using an optimal sol-gel/surface combination, which considers both signal variability and difference in signals between positive and negative controls, was determined to be 0.60, which is above the minimum level required for applicability to screening. By overprinting nanoliter volumes of solutions containing the dye, ATCh, and potential inhibitors, these microarrays could be used to screen two libraries of small molecules, one composed of newly synthesized alkaloids and another consisting of ∼1000 known bioactive compounds, both as discrete compounds and mixtures thereof, for activity against AChE. IC(50) values were obtained on microarrays for compounds showing significant inhibitory activity, demonstrating the utility of arrays for quantitative inhibition assays.

An ESI-MS/MS method for screening of small-molecule mixtures against glycogen synthase kinase-3beta (GSK-3beta).

Glycogen synthase kinase-3beta (GSK-3beta) is involved in the hyperphosphorylation of previously phosphorylated (primed) substrates, and is currently assayed using an approach based on the incorporation of gamma-(32)P-radiolabelled isotopes into substrate peptides. The requirement to detect hyperphosphorylation of a primed substrate poses a particular challenge for development of a high-throughput screening assay, as many current kinase assays are designed to produce a signal in the presence of any phosphorylation site, and thus are only suitable for beta-unphosphorylated substrates. Herein, we have developed an electrospray-ionization tandem mass spectrometry (ESI-MS/MS) assay to allow for direct detection of a hyperphosphorylated product which is formed in a solution reaction involving a primed peptide substrate (GSM peptide) and GSK-3beta. Optimum reaction conditions (level of Mg(2+), buffer type, ionic strength, pH, enzyme concentration, and reaction time) were established to both maintain the activity of GSK-3beta and allow for substrate and product quantification through ESI/MS/MS. We show that the MS-based assay allows for rapid determination of GSK-3beta activity from reaction volumes of approximately 40 microL and that it can be used to assess IC(50) values and the site of action of known inhibitors. It also can be used for automated screening of small-molecules mixtures to identify inhibitors of GSK-3beta.

Inhibitor screening using immobilized enzyme reactor chromatography/mass spectrometry.

We describe the coupling of capillary-scale monolithic enzyme reactor columns directly to a tandem mass spectrometer for screening of enzyme inhibitors. A two-channel nanoLC system is used to continuously infuse substrate or substrate/inhibitor mixtures through the column, allowing continuous variation of inhibitor concentration by simply altering the ratio of flow from the two pumps. In the absence of inhibitor, infusion of substrate leads to formation of product, and both substrate and product ions can be simultaneously monitored in a quantitative manner by MS/MS. The presence of inhibitor leads to a decrease in product and an increase in substrate concentration in the column eluent. Knowing the product/substrate ratio and the total analyte concentration (P + S), the concentration of product eluting, and hence the relative enzyme activity, can be determined. Both IC50 and KI values can then be obtained by direct MS detection of the effect of inhibitors on relative activity. Inhibitor screening is demonstrated using reusable, sol-gel derived, monolithic capillary columns containing adenosine deaminase, directly interfaced to ESI-MS/MS. On-column enzyme activity was assessed by monitoring inosine and adenosine elution. It is shown that the method can be used for automated screening of the effects of compound mixtures on ADA activity and to determine the KI value of the known inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, even when the compound is present within a mixture.

Entrapment of fluorescent signaling DNA aptamers in sol-gel-derived silica.

We report on the first successful immobilization of a DNA aptamer, in particular, a fluorescence-signaling DNA aptamer, within a sol-gel-derived matrix. The specific aptamer examined in this study undergoes a structural switch in the presence of adenosine triphosphate (ATP) to release a dabcyl-labeled nucleotide strand (QDNA), which in turn relieves the quenching of a fluorescein label that is also present in the aptamer structure. It was demonstrated that aptamers containing a complementary QDNA strand along with either a short complimentary strand bearing fluorescein (tripartite structure) or a directly bound fluorescein moiety (bipartite structure) remained intact upon entrapment within biocompatible sol-gel derived materials and retained binding activity, structure-switching capabilities, and fluorescence signal generation that was selective and sensitive to ATP concentration. Studies were undertaken to evaluate the properties of the immobilized aptamers that were either in their native state or bound to streptavidin using a terminal biotin group on the aptamer, including response time, accessibility, and leaching. Furthermore, signaling abilities were optimized through evaluation of different QDNA constructs. These studies indicated that the aptamers remained in a state that was similar to solution, with moderate leaching, only minor decreases in accessibility to ATP, and an expected reduction in response time due to diffusional barriers to mass transport of the analyte through the silica matrix. Entrapment of the aptamer also resulted in protection of the DNA against degradation from nucleases, improving the potential for use of the aptamer for in vivo sensing. This work demonstrates that sol-gel-derived materials can be used to successfully immobilize and protect DNA-based biorecognition elements and, in particular, DNA aptamers, opening new possibilities for the development of DNA aptamer-based devices, such as affinity columns, microarrays, and fiber-optic sensors.

Screening of inhibitors using enzymes entrapped in sol-gel-derived materials.

In recent years, a number of new methods have been reported that make use of immobilized enzymes either on microarrays or in bioaffinity columns for high-throughput screening of compound libraries. A key question that arises in such methods is whether immobilization may alter the intrinsic catalytic and inhibition constants of the enzyme. Herein, we examine how immobilization within sol-gel-derived materials affects the catalytic constant (kcat), Michaelis constant (KM), and inhibition constant (KI) of the clinically relevant enzymes Factor Xa, dihydrofolate reductase, cyclooxygenase-2, and gamma-glutamyl transpeptidase. These enzymes were encapsulated into sol-gel-derived glasses produced from either tetraethyl orthosilicate (TEOS) or the newly developed silica precursor diglyceryl silane (DGS). It was found that the catalytic efficiency and long-term stability of all enzymes were improved upon entrapment into DGS-derived materials relative to entrapment in TEOS-based glasses, likely owing to the liberation of the biocompatible reagent glycerol from DGS. The KM values of enzymes entrapped in DGS-derived materials were typically higher than those in solution, whereas upon entrapment, kcat values were generally lowered by a factor of 1.5-7 relative to the value in solution, indicating that substrate turnover was limited by partitioning effects or diffusion through the silica matrix. Nonetheless, the apparent KI value for the entrapped enzyme was in most cases within error of the value in solution, and even in the worst case, the values differed by no more than a factor of 3. The implications of these findings for high-throughput screening are discussed.