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Screening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves tedious steps such as affinity selection, bead picking, sequencing, and characterization. Herein, we present a high-throughput &ldquo, all-on-one chip&rdquo, system to avoid slow and technically complex bead picking steps. On a traditional glass slide provided with an electrically conductive tape, beads of a combinatorial peptide library are aligned and immobilized by application of a precision sieve. Subsequently, the chip is incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracy without picking of the positive beads. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to nearly 100%. The new technique was validated by employing a FLAG-based model system, identifying new peptide binders for the monoclonal M2 anti-FLAG antibody, and was finally utilized to search for IgG-binding peptides. In the present format, more than 30,000 beads can be screened on one slide.

Biosensors are nowadays ubiquitous in biomedical diagnosis as well as a wide range of other areas such as point-of-care monitoring of treatment and disease progression, environmental monitoring, food control, drug discovery, forensics and biomedical research. A wide range of techniques can be used for the development of biosensors. Their coupling with high-affinity biomolecules allows the sensitive and selective detection of a range of analytes. We give a general introduction to biosensors and biosensing technologies, including a brief historical overview, introducing key developments in the field and illustrating the breadth of biomolecular sensing strategies and the expansion of nanotechnological approaches that are now available.

Inositol-based signaling molecules are central eukaryotic messengers and include the highly phosphorylated, diffusible inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs). Despite the essential cellular regulatory functions of InsPs and PP-InsPs (including telomere maintenance, phosphate sensing, cell migration, and insulin secretion), the majority of their protein targets remain unknown. Here, the development of InsP and PP-InsP affinity reagents is described to comprehensively annotate the interactome of these messenger molecules. By using the reagents as bait, >150 putative protein targets were discovered from a eukaryotic cell lysate (Saccharomyces cerevisiae). Gene Ontology analysis of the binding partners revealed a significant overrepresentation of proteins involved in nucleotide metabolism, glucose metabolism, ribosome biogenesis, and phosphorylation-based signal transduction pathways. Notably, we isolated and characterized additional substrates of protein pyrophosphorylation, a unique posttranslational modification mediated by the PP-InsPs. Our findings not only demonstrate that the PP-InsPs provide a central line of communication between signaling and metabolic networks, but also highlight the unusual ability of these molecules to access two distinct modes of action.

For more than 30years, the production of research antibodies has been dominated by hybridoma technologies, while modern recombinant technologies have lagged behind. Here I discuss why this situation must change if we are to generate reliable, comprehensive reagent sets on a genome-wide scale, and I describe how a cultural shift in the research community could revolutionize and modernize the affinity reagent field. In turn, such a revolution would pay huge dividends by closing the gap between basic research and therapeutic development, thus enabling the development of myriad new therapies for unmet medical needs.

To enhance their therapeutic potential, human antibody heavy chain variable domains (VHs) would benefit from increased thermostability. The highly conserved disulfide linkage that connects Cys23 and Cys104 residues in the core of VH domains is crucial to their stability and function. It has previously been shown that the introduction of a second disulfide linkage can increase the thermostability of camelid heavy-chain antibody variable domains (VHHs). Using four model domains we demonstrate that this strategy is also applicable to human VH domains. The introduced disulfide linkage, formed between Cys54 and Cys78 residues, increased the thermostability of VHs by 1418°C. In addition, using a novel hexa-histidine capture technology, circular dichroism, turbidity, size exclusion chromatography and multiangle light scattering measurements, we demonstrate reduced VH aggregation in domains with the Cys54Cys78 disulfide linkage. However, we also found that the engineered disulfide linkage caused conformational changes, as indicated by reduced binding of the VHs to protein A. This indicates that it may be prudent to use the synthetic VH libraries harboring the engineered disulfide linkage before screening for affinity reagents. Such strategies may increase the number of thermostable binders. © 2012 The Author.

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

With the sequencing of an eukaryotic genome, it is possible to inventory the predicted proteome for proteins that carry one or more Src Homology 3 (SH3) domains. Due to the current ease of cloning and gene synthesis, these short domains can be readily overexpressed and manipulated for the purpose of characterizing their specificity and affinity for peptide ligands, as well as solving the three-dimensional structures of the domains. This information can be used to predict and confirm their cellular interacting partners, in the effort to understand the function of a eukaryotic protein by focusing on its SH3 domain. Finally, capitalizing on our mature understanding about protein–protein interacting modules, like the SH3 domain, it is possible to use directed evolution to enhance or change the specificity and affinity of an SH3 domain for the purpose of creating reagents to be used in biochemical purification or cell perturbation studies.