Combinatorial Ligand Libraries Research Articles

Specific Electrostatic Molecular Recognition in Water.

The identification of pairs of small peptides that recognize each other in water exclusively through electrostatic interactions is reported. The target peptide and a structure-biased combinatorial ligand library consisting of ≈78 125 compounds were synthesized on different sized beads. Peptide-peptide interactions could conveniently be observed by clustering of the small, fluorescently labeled target beads on the surface of larger ligand-containing beads. Sequences of isolated hits were determined by MS/MS. The interactions of the complex showing the highest affinity were investigated by a novel single-bead binding assay and by 2D NMR spectroscopy. Molecular dynamics (MD) studies revealed a putative mode of interaction for this unusual electrostatic binding event. High binding specificity occurred through a combination of topological matching and electrostatic and hydrogen-bond complementarities. From MD simulations binding also seemed to involve three tightly bound water molecules in the interface between the binding partners. Binding constants in the submicromolar range, useful for biomolecular adhesion and in nanostructure design, were measured.

Combinatorial ligand libraries as a two-dimensional method for proteome analysis

The present report tries to assess the possibility of performing capture of proteomes via combinatorial peptide ligand libraries (CPLL) in a two-dimensional (2D) mode, i.e. via orthogonal complementarity in the capture phase. To that aim, serum proteins are captured at physiological pH either at low ionic strength (25mM NaCl) or at high concentrations of lyotropic salts of the Hofmeister series (1M ammonium sulphate) favouring hydrophobic interaction. Indeed such 2D mechanisms seems to be operative, since 52% of the captured proteins are common to the two capture modes, 20% are specific only of the “ionic” interaction mode and 28% are found only in the “hydrophobically” driven interaction. As an additional bonus, losses of protein species from the initial sample, one of the major drawbacks of CPLLs, are diminished to about 5% and are found only in the ionic capture, whereas the hydrophobically engendered capture is loss-free.

The art of observing rare protein species in proteomes with peptide ligand libraries

The present review deals with the use of combinatorial ligand libraries, composed by hexapeptides, in the capture and concentration of the low-abundance proteome. This method, first reported in 2005, is compared with other current methodologies aimed at exploring the "deep proteome", such as: depletion of high-abundance proteins (especially in sera and cerebrospinal fluid) by individual or combined antibodies (up to 20 against the most abundant species); narrow (1-pH-unit) IPGs (zoom gels) and prefractionation with multicompartment electrolyzers or with Off-Gel electrophoresis. The physico-chemical properties of the hexapeptide library are also explored, namely in assessing the proper length of the baits and the behavior of shorter oligopeptides, down to capture elicited by single amino acids. A number of examples on the use of this library is given, such as the analysis of biological fluids (human sera, urine, bile, cerebrospinal fluid) and of cell lysates (platelets, red blood cells). In all cases, it was possible to detected from three to five times as many proteins as compared to control, untreated samples. Perhaps the most spectacular results were obtained with the erythrocyte proteome, where 1570 proteins could be identified in the "minority" proteome, representing only 2% of the total cell lysate. Another interesting area of application regards the concentration and detection of trace impurities contaminating r-DNA proteins meant for human consumption: several host proteins, never reported before, could be revealed for the first time. Other nonhuman samples are currently under investigation, such as egg-white (where no less than 148 unique gene products could be identified), egg yolk (with 255 unique species) and latex from Hevea brasiliensis. It is anticipated that the ligand library could be a most useful tool for detecting biomarkers for different pathologies and for drug treatment. As a future outlook, one could envision the synthesis of specialized libraries able to capture given classes of proteins (e.g., phospho- and glyco-proteins). Additionally, the library could be used in association with other techniques currently in vogue, such as zoom gels, Off-Gels, and the like.

The ProteoMiner in the proteomic arena: A non-depleting tool for discovering low-abundance species

Combinatorial ligand libraries, composed by millions of hexapeptides, are here reviewed in terms of their ability of capturing the low-abundance proteome. First, the physico-chemical properties of such libraries are dealt with, especially in regard to the proper length of the bait. The capturing ability of single amino acids has been assessed demonstrating that there exist a protein adsorption capability dichotomy, by which 8 amino acids (Arg, Lys, His, Phe, Tyr, Trp, Val and Leu) are classified as interacting with a large number of proteins with all the remaining amino acids with limited capturing capabilities. The highest performance in capturing the largest possible population of proteins is offered by the three hydrophobic, aromatic amino acids, i.e. Phe, Tyr and Trp, suggesting that hydrophobic motifs are those responsible for the strongest, and most frequently occurring, interactions. By exploring baits ranging from single, individual amino acids, to di-, tri-, tetra- penta- and hexapeptides, it was demonstrated that the 6-mer baits are the ones with the most promising length for capturing the largest possible population of proteins and that probably longer lengths would hardly be needed. Some examples are given on the ability to explore the low-abundance proteome in two systems, notably chicken egg white and yolk. In both cases, by using the peptide library methodology, it is possible to detect at least twice as many protein species as compared to the best results obtained so far with the most advanced proteomics studies using highly sophisticated mass spectrometry tools.

Immunoassays: Biological Tools for High Throughput Screening and Characterisation of Combinatorial Libraries

In the demanding field of proteomics, there is an urgent need for affinity-catcher molecules to implement effective and high throughput methods for analysing the human proteome or parts of it. Antibodies have an essential role in this endeavour, and selection, isolation and characterisation of specific antibodies represent a key issue to meet success. Alternatively, it is expected that new, well-characterised affinity reagents generated in rapid and cost-effective manners will also be used to facilitate the deciphering of the function, location and interactions of the high number of encoded protein products. Combinatorial approaches combined with high throughput screening (HTS) technologies have become essential for the generation and identification of robust affinity reagents from biological combinatorial libraries and the lead discovery of active/mimic molecules in large chemical libraries. Phage and yeast display provide the means for engineering a multitude of antibody-like molecules against any desired antigen. The construction of peptide libraries is commonly used for the identification and characterisation of ligand-receptor specific interactions, and the search for novel ligands for protein purification. Further improvement of chemical and biological resistance of affinity ligands encouraged the "intelligent" design and synthesis of chemical libraries of low-molecular-weight bio-inspired mimic compounds. No matter what the ligand source, selection and characterisation of leads is a most relevant task. Immunological assays, in microtiter plates, biosensors or microarrays, are a biological tool of inestimable value for the iterative screening of combinatorial ligand libraries for tailored specificities, and improved affinities. Particularly, enzyme-linked immunosorbent assays are frequently the method of choice in a large number of screening strategies, for both biological and chemical libraries.

Hexapeptide Combinatorial Ligand Libraries: The March for the Detection of the Low-Abundance Proteome Continues

After 10 years of extensive proteomic research, it has become increasingly apparent that new technologies are sorely needed for detecting the low-abundance proteome-those proteins (up to 50% in any proteome) whose concentration in tissues or cells falls below the detection limits of currently available methodologies. Here we survey one such method: a combinatorial ligand library (called ProteoMiner), comprising dozens of millions of hexapeptides capable of interacting with most, if not all, proteins in any given proteome. They act by drastically reducing the signal of high-abundance species while increasing the level of the low-abundance components to bring their signal within the detection limit of present-day tools. Such a library has been tested against a number of human biological fluids, such as sera, urine, cerebrospinal fluid as well as against cell lysates (e.g., platelets, red blood cells) with interesting results.

Proteomics and transfusion medicine.

Proteomics is becoming an important research tool which, together with genomics, will, in the near future, presumably have an impact on disease diagnosis and prognosis as well as on further advances in the production and storage processes of blood-based therapeutics.

The “Invisible Proteome”: How to Capture the Low-Abundance Proteins Via Combinatorial Ligand Libraries

Despite recent advances in pre-fractionation procedures and depletion methods via immuno-subtraction protocols of the most abundant species, the “low-abundance” proteome remains largely undetected. We report here a novel technology, called ProteoMiner, for bringing to the limelight this vast pool of low-abundance species, which could constitute > 50% of any proteome in any living organism. It consists of a combinatorial ligand library, composed of millions of diverse hexapeptide baits, able to capture and concentrate the “low-abundance” proteome, while drastically cutting the concentration of the most abundant compounds. Since no depletion of any species is contemplated by this methodology, but a drastic reduction of the sample dynamic range, the noxious phenomenon of co-depletion (especially troublesome in affinity depletion methods) does not occur. In addition to previously reported data on analysis of human sera and urines, we describe here novel applications for the detection of the low-abundance proteome in human blood cells, such as the red blood cells (RBC) and platelets and in biological fluids, such as cerebrospinal fluid. In particular, in the case of RBC, where hemoglobin alone constitutes ca. 98% of the total cytoplasmic proteome, the ProteoMiner technology allowed the detection of > 1500 proteins in the remaining 2% low-abundance cytoplasmic proteome, most of them not previously reported even in the most advanced investigations. The merits and limits of ProteMiner are discussed and evaluated.

Exploring the Platelet Proteome via Combinatorial, Hexapeptide Ligand Libraries

A combinatorial ligand library, composed of millions of diverse hexapeptide baits, able to capture and concentrate the "low-abundance" proteome while drastically cutting the concentration of the most abundant species, has been applied to the exploration of the soluble platelet proteome. Mass spectrometry analysis of untreated and library-treated platelets has resulted in the identification of 435 unique gene products. Of those, 147 entries (35% of the total) have not been described among the list of >1100 proteins in proteomic platelet investigations reported before. In addition, the analysis of excised spots from two-dimensional electrophoresis analysis allowed 57 other proteins to be added that were not found in LC-MS analysis, 33 of them not described before in proteomics studies, bringing the total number of new gene products to 180. Thus, the present data add a non-negligible number of species for continuing the "cartography" of the proteomic asset of platelets, in view of completing the mapping procedure for a deeper understanding of the physiology and pathology of this blood cell. Because the capturing process is performed under physiological conditions, by exploiting, for binding to the combinatorial library, the native protein configuration, the described technique is not adapted to capture highly hydrophobic proteins, which need strong denaturing and solubilizing conditions that are incompatible with our working procedure. Thus, our list reports essentially hydrophilic proteins, with negative GRAVY indexes.

Investigation of disulfonamide ligands derived from o-phenylenediamine and their Pb(II) complexes by electrospray ionization mass spectrometry.

An electrospray ionization mass spectrometry (ESI-MS) method, in both positive and negative ion modes, was developed for characterization of disulfonamide ligands derived from o-phenylenediamine and their Pb(II) complexes. For the ligands, negative ion mode ESI-MS in methanolic solutions gave simple and easily interpretable mass spectra. However, the spectra of Pb complexes were not readily interpretable under the same conditions. Protonated ligands and their Pb(II) complexes were observed in methanolic solutions by ESI-MS in positive ion mode. The formation of Na(+), K(+), or NH(4) (+) adducts was also observed, complicating the mass spectra and decreasing the signal intensity. In order to optimize the detection of the ligands and the Pb complexes, a method was developed by adding NaOAc in the solutions. The presence of 0.2 mM NaOAc simplified the mass spectra of the ligands and the Pb complexes, and significantly increased sensitivity in both negative and positive ion modes. This modification makes ESI-MS in both modes suitable for characterization of sulfonamide ligands and their Pb complexes, thus providing a potentially powerful tool for evaluating formation of metal complexes and screening combinatorial ligand libraries.