The Bead blot: a method for selecting small molecule ligands for protein capture and purification

We present a new method for selecting peptide ligands that are useful for protein purification, protein targeting and exploring protein-ligand interactions, and which requires no prior protein purification or derivatization. In the Bead blot, a complex mixture containing the target protein, for example, plasma, is incubated with a combinatorial library of peptide ligands synthesized on beads. The proteins are fractionated and purified on their respective ligands and the beads with their bound proteins are immobilized in a gel. The proteins are eluted from the ligands by capillary action and captured on a membrane so that their position on the membrane corresponds to the position of the beads in the gel. The protein is detected on the membrane, generating spots on an autoradiography film, the spots on the film are aligned with the beads in the gel, and the beads that bound the protein are recovered. The ligand on the bead(s) can be sequenced and synthesized at large scale for protein purification. The Bead blot can be completed in several hours with overnight pause steps if desired. On average, 5 prospective ligands are selected per 50,000 beads screened using this method. Unlike other ligand identification methods, the target protein does not have to be purified or labeled, and the Bead blot allows ligands for multiple proteins to be selected in a single experiment.     

The Bead blot: a method for identifying ligand-protein and protein-protein interactions using combinatorial libraries of peptide ligands

Small molecules that bind proteins can be used as ligands for protein purification and for investigating protein-protein and protein-drug interactions. Unfortunately, many methods used to identify new ligands to desired proteins suffer from common shortcomings, including the requirement that the target protein be purified and/or the requirement that the ligands be selected under conditions different from those under which it will be used. We have developed a new method called the Bead blot that can (i) select ligands to unpurified proteins, including trace proteins, present in complex materials (e.g., unfractionated plasma); (ii) select ligands to multiple proteins under a variety of conditions in a single experiment; and (iii) be used with libraries of different types of ligands. In the Bead blot, a library of ligands, synthesized on chromatography resin beads, is incubated with a starting material containing a target protein for which a ligand is sought. The proteins in the material bind to their complementary ligands according to specific affinity interactions. Then the protein-loaded beads are immobilized in a porous matrix, and the proteins are directionally eluted from the beads and captured on a membrane superimposed on the beads. The location of the target protein on the membrane is determined, and because the position of the protein(s) on the membrane reflects the position of the bead(s) in the matrix, the bead that originally bound the protein is identified, with subsequent elucidation of the ligand sequence. Ligands to several targets can be identified in one experiment. Here we demonstrate the broad utility of this method by the selection of ligands that purify plasma protein complexes or that remove pathogens from whole blood with very high affinity constants. We also select ligands to a protein based on competitive elution.     

Selective ligand purification using high-performance affinity beads

Since the development of affinity chromatography, affinity purification technology has been applied to many aspects of biological research, becoming an indispensable tool. Efficient strategies for the identification of biologically active compounds based on biochemical specificity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that specifically interact with a target biomolecule using reverse affinity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was efficiently purified in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse affinity beads facilitated identification of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were purified from a crude cell extract, respectively. These data indicate that reverse affinity beads are powerful tools for identification of both specific ligands and proteins that interact with receptor/ligand complexes.     

Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach

Shotgun proteomics protocols are widely used for the identification and/or quantitation of proteins in complex biological samples. Described here is a shotgun proteomics protocol that can be used to identify the protein targets of biologically relevant ligands in complex protein mixtures. The protocol combines a quantitative proteomics platform with a covalent modification strategy, termed Stability of Proteins from Rates of Oxidation (SPROX), which utilizes the denaturant dependence of hydrogen peroxide-mediated oxidation of methionine side chains in proteins to assess the thermodynamic properties of proteins and protein-ligand complexes. The quantitative proteomics platform involves the use of isobaric mass tags and a methionine-containing peptide enhancement strategy. The protocol is evaluated in a ligand binding experiment designed to identify the proteins in a yeast cell lysate that bind the well-known enzyme cofactor, β-nicotinamide adenine dinucleotide (NAD+). The protocol is also used to investigate the protein targets of resveratrol, a biologically active ligand with less well-understood protein targets. A known protein target of resveratrol, cytosolic aldehyde dehydrogenase, was identified in addition to six other potential new proteins targets including four that are associated with the protein translation machinery, which has previously been implicated as a target of resveratrol.     

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.     

Proteomic analysis of human blood serum using peptide library beads

Human serum is thought to contain key information for diagnostics of human disease. However, no single technology is currently nor might ever be available to cope with the complexity and dynamic range of the serum proteome. We here report a large-scale proteomic study of human blood serum using peptide library beads and mass spectrometry. Serum proteins are adsorbed onto polymeric beads coated with a combinatorial library composed of millions of hexameric peptide baits. Analysis of the eluates from this combinatorial library (as obtained with 3 eluants of different strength, able to release 99% of the retentate) via liquid chromatography coupled to high-resolution mass spectrometry resulted in the identification of 1559 proteins or 3869 proteins, respectively, depending on how 95% confidence was estimated. In either case, the analysis showed that ligand beads are able to capture a large number of proteins in a single operation. The ligand bead bound fraction appeared to have a lower dynamic range when compared to the starting material, due to a "normalization" of the protein concentrations in the original mixture. We find that extensive information on the protein composition of complex samples such as serum can be obtained using ligand beads and that these beads enrich the proteomic tool box.     

The scaffolding protein CG-NAP/AKAP450 is a critical integrating component of the LFA-1-induced signaling complex in migratory T cells

T cell migration represents a complex highly coordinated process involving participation of surface receptor/ligand interactions, cytoskeletal rearrangements, and phosphorylation-dependent signaling cascades. Members of the A-kinase anchoring protein (AKAP) family of giant scaffolding proteins can assemble and compartmentalize multiple signaling and structural molecules thereby providing a platform for their targeted positioning and efficient interactions. We characterize here the expression, intracellular distribution, and functional role of the scaffolding protein CG-NAP (centrosome and Golgi localized protein kinase N-associated protein)/AKAP450 in the process of active T cell motility induced via LFA-1 integrins. This protein is predominantly localized at the centrosome and Golgi complex. T cell locomotion triggered by LFA-1 ligation induces redistribution of CG-NAP/AKAP450 along microtubules in trailing cell extensions. Using an original in situ immunoprecipitation approach, we show that CG-NAP/AKAP450 is physically associated with LFA-1 in the multimolecular signaling complex also including tubulin and the protein kinase C beta and delta isoenzymes. CG-NAP/AKAP450 recruitment to this complex was specific for the T cells migrating on LFA-1 ligands, but not on the beta(1) integrin ligand fibronectin. Using the GFP-tagged C-terminal CG-NAP/AKAP450 construct, we demonstrate that expression of the intact CG-NAP/AKAP450 and its recruitment to the LFA-1-associated multimolecular complex is critically important for polarization and migration of T cells induced by this integrin.     

Reconstitution and purification of cytolethal distending toxin of Actinobacillus actinomycetemcomitans

Cytolethal distending toxin (CDT) has been found in various pathogenic bacterial species and causes a cell distending and a G2 arrest against eukaryotic cells. All the cdtABC genes, which encode CDT, are known to be required for the CDT activities although the CDT holotoxin structure has not been elucidated. We cloned the cdtABC genes of Actinobacillus actinomycetemcomitans and constructed an Escherichia coli expression system for them. We found that crude extracts from six deletion mutants (delta cdtA, delta cdtB, delta cdtC, delta cdtBC, delta cdtAC, and delta cdtAB) of recombinant E. coli, which showed very weak or no detectable CDT activities, restored the CDT activities when pre-mixing and pre-incubation of them were performed in combinations to contain all the CdtA, CdtB, and CdtC proteins. These results indicate that all the Cdt proteins are required for the CDT activities. We also found that the chimera CdtB protein, CdtB-intein-CBD (chitin binding domain) like CdtB protein itself assembled with CdtA and CdtC. The reconstituted CDT containing the chimera CdtB protein was specifically extracted by chitin beads and the only CDT portion was isolated from the chitin beads by a cleavage reaction of the intein. The purified reconstituted-CDT was found to consist of CdtA, CdtB, and CdtC proteins, and showed appreciable CDT activities, indicating that the CDT holotoxin structure is the CdtABC complex. To our knowledge, this is the first report succeeded in complete purification of an active CDT and may offer useful tools for elucidation of the toxic mechanism of CDT.     

Crystal structure of BMP-9 and functional interactions with pro-region and receptors

Bone morphogenetic proteins (BMPs), a subset of the transforming growth factor (TGF)-beta superfamily, regulate a diverse array of cellular functions during development and in the adult. BMP-9 (also known as growth and differentiation factor (GDF)-2) potently induces osteogenesis and chondrogenesis, has been implicated in the differentiation of cholinergic neurons, and may help regulate glucose metabolism. We have determined the structure of BMP-9 to 2.3 A and examined the differences between our model and existing crystal structures of other BMPs, both in isolation and in complex with their receptors. TGF-beta ligands are translated as precursors, with pro-regions that generally dissociate after cleavage from the ligand, but in some cases (including GDF-8 and TGF-beta1, -2, and -3), the pro-region remains associated after secretion from the cell and inhibits binding of the ligand to its receptor. Although the proregion of BMP-9 remains tightly associated after secretion, we find, in several cell-based assays, that the activities of BMP-9 and BMP-9.pro-region complex were equivalent. Activin receptor-like kinase 1 (ALK-1), an orphan receptor in the TGF-beta family, was also identified as a potential receptor for BMP-9 based on surface plasmon resonance studies (BIAcore) and the ability of soluble ALK-1 to block the activity of BMP-9.pro-region complex in cell-based assays.     

Ligands selected from combinatorial libraries of protein A for use in affinity capture of apolipoprotein A-1M and taq DNA polymerase

Here we show that robust and small protein ligands can be used for affinity capture of recombinant proteins from crude cell lysates. Two ligands selectively binding to bacterial Taq DNA polymerase and human apolipoprotein A-1(M), respectively, were used in the study. The ligands were selected from libraries of a randomized alpha-helical bacterial receptor domain derived from staphylococcal protein A and have dissociation constants in the micromolar range, which is typical after primary selection from these libraries consisting of approximately 40 million different members each. Using these ligands in affinity chromatography, both target proteins were efficiently recovered from crude cell lysates with high selectivities. No loss of column capacity or selectivity was observed for repeated cycles of sample loading, washing and low pH elution. Interestingly, column sanitation could be performed using 0. 5 M sodium hydroxide without significant loss of ligand performance. The results suggest that combinatorial approaches using robust protein domains as scaffolds can be a general tool in the process of designing purification strategies for biomolecules.     

Assay development and data analysis of receptor-ligand binding based on scintillation proximity assay

In this paper, we described the optimization of a generic binding assay to measure ligand-receptor interactions for peroxisome proliferator-activated receptors (PPARs). The assay is based on scintillation proximity assay, in which a protein is coated on scintillant-incorporated beads, and a radiolabeled ligand stimulates the beads to emit a signal by binding to the immobilized protein. An intrinsic binding affinity of unlabeled ligands is determined by competitive displacement of the radioligand. The protein coating and ligand binding are achieved in one step by simply mixing ligands, protein and beads in sequence. No additional steps of pre-coating and washing of beads are required. Protein is captured on beads effectively by electrostatic interactions, thus no affinity labeling of protein is required. In data analysis, ligands are grouped into two classes based on their binding affinities. For tight binding ligands, an equation is derived to accurately determine the binding affinity. Otherwise a general equation applies. This quantitative and high throughput assay provides a tool to screen a large library of molecules in search of potent ligands.     

Pre‐fractionation of rat liver cytosol proteins prior to mass spectrometry‐based proteomic analysis using tandem biomimetic affinity chromatography

Efficient and high resolution separation of the protein mixture prior to trypsin digestion and mass spectrometry (MS) analysis is generally used to reduce the complexity of samples, an approach that highly increases the probability of detecting low‐copy‐number proteins. Our laboratory has constructed an affinity ligand library composed of thousands of ligands with different protein absorbance effects. Structural differences between these ligands result in different non‐bonded protein–ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first selected out several synthetic affinity ligands showing large band distribution differences in proteins absorbance profiles, and a tandem composition of these affinity ligands was used to distribute complex rat liver cytosol into simple subgroups. Ultimately, all the fractions collected from tandem affinity pre‐fractionation were digested and then analyzed by LC‐MS/MS, which resulted in high confidence identification of 665 unique rat protein groups, 1.8 times as many proteins as were detected in the un‐fractionated sample (371 protein groups). Of these, 375 new proteins were identified in tandem fractions, and most of the proteins identified in un‐fractionated sample (290, 80%) also emerged in tandem fractions. Most importantly, 430 unique proteins (64.7%) only characterized in specific fractions, indicating that the crude tissue extract was well distributed by tandem affinity fractionation. All detected proteins were bioinformatically annotated according to their physicochemical characteristics (such as MW, pI, GRAVY value, TM Helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes. Combined usage of tandem affinity pre‐fractionation with MS‐based proteomic analysis is simple, low‐cost, and effective, providing the prospect of broad application in proteomics. Copyright © 2009 John Wiley & Sons, Ltd.     

Equalizer technology – Equal rights for disparate beads

One major limitation in proteomics is the detection and analysis of low‐abundant proteins, i.e. in plasma. Several years ago, a technique to selectively enrich the relative concentration of low‐abundant proteins was introduced by Boschetti and co‐workers. It is based on a specific and saturable interaction of proteins to a high diversity of binding sites, realized by a hexapeptide library coupled to beads. This technology was commercialized as Equalizer beads or ProteoMiner. However, during application of ProteoMiner beads to plasma samples unexpected results questioned the proposed mode of action. Therefore, ProteoMiner beads were compared with chromatographic beads exhibiting completely different surface chemistry. Sepabeads FP‐OD400 octadecyl, FP‐DA400 diethylamine, FP‐BU400 butyl, FP‐HG400 hydroxyl and EXE056 epoxy were used. The results show that ProteoMiner or the different Sepabeads behave surprisingly similarly in the separation of complex protein mixtures. ProteoMiner beads interact with protein mixtures according to a general hydrophobic binding mechanism, where diversity in surface ligands plays only a negligible role.     

Activity-based in vitro selection of T4 DNA ligase

Recent in vitro methodologies for selection and directed evolution of proteins have concentrated not only on proteins with affinity such as single-chain antibody but also on enzymes. We developed a display technology for selection of T4 DNA ligase on ribosome because an in vitro selection method for DNA ligase had never been developed. The 3' end of mRNA encoding the gene of active or inactive T4 DNA ligase-spacer peptide fusion protein was hybridized to dsDNA fragments with cohesive ends, the substrate of T4 DNA ligase. After in vitro translation of the mRNA-dsDNA complex in a rabbit reticulocyte system, a mRNA-dsDNA-ribosome-ligase complex was produced. T4 DNA ligase enzyme displayed on a ribosome, through addition of a spacer peptide, is able to react with dsDNA in the complex. The complex expressing active ligase was biotinylated by ligation with another biotinylated dsDNA probe and selected with streptavidin-coated magnetic beads. We effectively selected active T4 DNA ligase from a small amount of protein. The gene of the active T4 DNA ligase was enriched 40 times from a mixture of active and inactive genes using this selection strategy. This ribosomal display strategy may have high potential to be useful for selection of other enzymes associated with DNA.     

Affinity of mouse immunoglobulin G subclasses for sialic acid derivatives immobilized on dextran-coated supports

High-performance liquid affinity chromatography is a powerful method for the purification of biological compounds owing to its specificity, rapidity and high resolution. In our laboratory, we develop chromatographic supports based on porous silica beads. However, in order to minimize non-specific interactions between the inorganic surface and proteins in aqueous solution, the silica beads are coated with modified dextran. As previously reported, many affinity ligands can be covalently grafted onto dextran-coated silica. In this study, N-acetylneuramic acid, which belongs to the sialic acid family and is present in immunoglobulin G (IgG) epitopes, is used as an active ligand. The interactions of this affinity support and IgG subclasses are analyzed. This immobilized ligand enables purification of IgG3 antibodies.     

SpeedScreen: label-free liquid chromatography-mass spectrometry-based high-throughput screening for the discovery of orphan protein ligands

A high-throughput screening methodology tailored to the discovery of ligands for known and orphan proteins is presented. With this method, labeling of neither target protein nor screened compounds is required, as the ligands are affinity selected by incubation of the protein with mixtures of compounds in aqueous binding buffer. Unbound small-molecular-weight compounds are removed from the target protein:ligand complex by rapid size-exclusion chromatography in the 96-well format. The protein fraction is analyzed subsequently by liquid chromatography-mass spectrometry for detection and identification of the bound ligand. This screening method was validated with known protein:ligand model systems and optimized for selection of high-affinity binders in an industrial screening environment. All sample handling steps and the analytics are rapid, robust, and largely automated, adopting this technology to the needs of present high-throughput screening processes. This affinity-selection technology, termed SpeedScreen, is currently an integral part of our lead discovery process.     

The Utility of FK506-Binding Protein as a Fusion Partner in Scintillation Proximity Assays: Application to SH2 Domains

Methodology has been developed which gives a specific measure of the interaction of an SH2 domain with a phosphopeptide ligand using scintillation proximity assay (SPA) technology. Recombinant SH2 domains were expressed from a T7 RNA polymerase-based vector inEscherichia colias fusions to the C-terminus of the FK506-binding protein (FKBP) and purified from freeze-thaw lysates in high yield by affinity chromatography using immobilized phosphopeptides. For binding assays the phosphopeptide ligands were synthesized with a biotin tag and the FKBP fusion proteins were noncovalently radiolabeled with commercially available [³H]dihydroFK506. Complexes of tritiated SH2 fusion protein and biotinyl-phosphopeptide were then captured on streptavidin-coated SPA beads and counted. The modular protocol is an equilibrium technique that does not employ washing steps or specialized radiochemical syntheses required in other binding assays. The utility of the assay has been demonstrated in an examination of the ligand specificity of the SH2 domains of the tyrosine kinases ZAP70, Syk, and Lck. The methodology is potentially generalizable to any receptor–ligand interaction in which one component can be expressed as a fusion partner with FKBP and the other component can be captured on a SPA bead.     

Evaluation of the combination of bead technology with SELDI-TOF-MS and 2-D DIGE for detection of plasma proteins

Today biomarker discovery is one of the most active aspects of proteomic investigations. However, the wide dynamic range of plasma proteins makes the analysis very challenging because high abundance proteins tend to mask those of lower abundance. Using a large bead-based library of combinatorial peptide ligands (Equalizer beads or ProteoMiner), the dynamic range of the protein concentration is compressed, the high abundance proteins present in the sample are reduced and the low abundance proteins are enriched, while retaining representatives of all proteins within the sample. In the present study, the combination of beads with surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and two-dimensional differential gel electrophoresis (2-D DIGE) technology were evaluated considering efficiency, reproducibility, sensitivity, and compatibility. The bead technology is easily compatible with both SELDI-TOF-MS and 2-D DIGE and the samples can be analyzed directly without any processing of the sample. The use of the beads prior SELDI-TOF-MS and 2-D DIGE enabled detection of many new protein spots/peaks and increased resolution and improved intensity of low abundance proteins in a reproducible fashion compared with the depletion technique. Several proteins have been identified by the combination of beads, 2-D DIGE and MS for example different kinds of complement factors and cytoskeletal proteins. Our data suggest that integration of the bead technology with our current proteomic technologies will enhance the possibility to deliver new peptide/protein biomarker candidates in our projects.     

Design of novel lead molecules against RhoG protein as cancer target – a computational study

Cancer is a class of diseases characterized by uncontrolled cell growth. Every year more than 2 million people are affected by the disease. Rho family proteins are actively involved in cytoskeleton regulation. Over-expression of Rho family proteins show oncogenic activity and promote cancer progression. In the present work RhoG protein is considered as novel target of cancer. It is a member of Rho family and Rac subfamily protein, which plays pivotal role in regulation of microtubule formation, cell migration and contributes in cancer progression. In order to understand the binding interaction between RhoG protein and the DH domain of Ephexin-4 protein, the 3D structure of RhoG was evaluated and Molecular Dynamic Simulations was performed to stabilize the structure. The 3D structure of RhoG protein was validated and active site identified using standard computational protocols. Protein–protein docking of RhoG with Ephexin-4 was done to understand binding interactions and the active site structure. Virtual screening was carried out with ligand databases against the active site of RhoG protein. The efficiency of virtual screening is analysed with enrichment factor and area under curve values. The binding free energy of docked complexes was calculated using prime MM-GBSA module. The SASA, FOSA, FISA, PISA and PSA values of ligands were carried out. New ligands with high docking score, glide energy and acceptable ADME properties were prioritized as potential inhibitors of RhoG protein.