Physical elution in phage display selection of inorganic-binding peptides

Phage display is a commonly utilized in vivo approach in selecting peptides specific to solid inorganic materials. In this process, traditionally, high affinity peptides are recovered by a chemical elution method, which involves contacting the phage library with the desired inorganic, washing the weak binders, and eluting the tight binders under harsh buffer conditions. This process may result in incomplete removal of all strong binders, separation of the phage from the display protein, or may modify the material surface. To overcome these potential limitations, we developed a physical elution technique based on ultrasonication. Here, we report two optimized ultrasonication protocols by which we selected peptides specific to natural mineral mica. We first performed a 30-s physical elution after the chemical elution step and increased the efficiency of screening strong binders by about 100%. Encouraged by the results, we applied physical elution-only protocol where we obtained 45% of the selected sequences as strong binders. The approach has a far shorter total elution time, i.e., seconds compared to hours in traditional chemical elution. The novel physical elution approach using ultrasonication reported herein can be a highly efficient alternate step in the screening of solid material specific peptides.     

Identification of chiral selectors from a 200-member parallel combinatorial library

Selection of chiral selectors for the resolution of racemic N-(1-naphthyl)leucine ester 1 was studied with a 200-member parallel library prepared on polymeric synthesis resin. Through this study, the library screening procedure developed previously is improved and other pertinent issues concerning the screening process are also addressed. The equilibration time required for screening is reduced from 24 h to approximately 3 h. Excellent correlation of the outcome between the resin batch equilibration experiment and chromatographic separation is further demonstrated. It is also demonstrated that selectors with separation factors as low as 1.4 could be identified by this batch screening process. In addition, a great deal of information regarding enantioselective interactions was obtained in this parallel library study. Such information should prove useful in improving future library designs.     

Resolving isomeric peptide mixtures: a combined HPLC/ion mobility-TOFMS analysis of a 4000-component combinatorial library.

A reversed-phase high-performance liquid chromatography (HPLC) separation approach has been combined with ion mobility/time-of-flight (TOF) mass spectrometry in order to characterize a combinatorial peptide library designed to contain 4000 peptides of the general form NH2-Xxx-Xxx-XXX-CO2H, NH2-Ala-Xxx-Xxx-Xxx-CO2H, NH2-Ser-Ala-Xxx-Xxx-Xxx-CO2H and NH2-Leu-Ser-Ala-Xxx-Xxx-Xxx-CO2H (where Xxx represents a randomization over 10 different amino acids: Ala, Arg, Asp, Glu, Gly, Leu, Lys, Phe, Ser, and Val). Addition of the gas-phase mobility separation between the HPLC separation and TOF measurement dimensions makes it possible to resolve many peptide isomers that have identical retention times (and masses).     

Polyclonal antibody-based noncompetitive immunoassay for small analytes developed with short peptide loops isolated from phage libraries

To date, there are a few technologies for the development of noncompetitive immunoassays for small molecules, the most common of which relies on the use of anti-immunocomplex antibodies. This approach is laborious, case specific, and relies upon monoclonal antibody technology for its implementation. We recently demonstrated that, in the case of monoclonal antibody-based immunoassays, short peptide loops isolated from phage display libraries can be used as substitutes of the anti-immunocomplex antibodies for noncompetitive immunodetection of small molecules. The aim of this work was to demonstrate that such phage ligands can be isolated even when the selector antibodies are polyclonal in nature. Using phenoxybenzoic acid (PBA), a major pyrethroid metabolite, as a model system, we isolated the CFNGKDWLYC peptide after panning a cyclic peptide library on the PBA/anti-PBA immunocomplex. The sensitivity of the noncompetitive enzyme-linked immunosorbent assay (ELISA) setup with this peptide was 5-fold (heterologous) or 400-fold (homologous) higher than that of the competitive assay setup with the same antibody. Phage anti-immunocomplex assay (PHAIA) was also easily adapted into a rapid and highly sensitive dipstick assay. The method not only provides a positive readout but also constitutes a major shortcut in the development of sensitive polyclonal-based assays, avoiding the need of synthesizing heterologous competing haptens.     

Screening of a parallel combinatorial library for selectors for chiral chromatography

A method to screen parallel combinatorial libraries for chiral selectors is described. Crucial elements to such an approach are the efficient syntheses of potential chiral selectors on synthesis resins and the rapid screening of selectors attached to these resins with circular dichroism measurement. The method does not require any immobilization of the analyte and could complement the mixture combinatorial library method developed earlier in this laboratory. The feasibility of this parallel library screening procedure is demonstrated with a model study of the chiral HPLC resolution of (1-naphthyl)leucine ester (1) using a 16-member small library.     

Recognition of epoxy with phage displayed peptides

The development of a general approach for non-destructive chemical and biological functionalization of epoxy could expand opportunities for both fundamental studies and creating various device platforms. Epoxy shows unique electrical, mechanical, chemical and biological compatibility and has been widely used for fabricating a variety of devices. Phage display has emerged as a powerful method for selecting peptides that possess enhanced selectivity and binding affinity toward a variety of targets. In this letter, we demonstrate for the first time a powerful yet benign approach for identifying binding motifs to epoxy via comprehensively screened phage displayed peptides. Our results show that the epoxy can be selectively recognized with peptide-displaying phages. Further, along with the development of epoxy-based microstructures; recognition of the epoxy with phage displayed peptides can be specifically localized in these microstructures. We anticipate that these results could open up exciting opportunities in the use of peptide-recognized epoxy in fundamental biochemical recognition studies, as well as in applications ranging from analytical devices, hybrid materials, surface and interface, to cell biology.     

Resolution of tert-butyl-1-(2-methyinaphthyl)phosphine oxide using selectors identified from a chemical combinatorial library

Resolution of racemic tert-butyl-1-(2-methylnaphthyl)-phosphine oxide 1, a chiral phosphorus compound, was achieved using selectors developed from a small peptide library. Separation factors as high as 3.2 were observed. The library consists of 81 peptide-based potential chiral selectors on polymeric synthesis resins. The linker needed to immobilize the identified chiral selectors onto silica gel proved important in the chiral separation; a longer linker provided a significantly higher separation factor in this study.     

从噬菌体抗体库中筛选出抗原结合活性相近、表达水平不同的单链抗体

从抗人膀胱癌改形噬菌体抗体库中筛选出一组相对亲和力常数和细胞结合活性相近的单链抗体，而它们的结合活性均明显高于起始的鼠源单链抗体。在亲和力淘选的过程中，这一组抗体的表达量存在显著差异，这在一定程度上影响到筛选过程中ELISA鉴定的结果，使整个筛选过程并不仅仅是对高亲和力抗体的筛选，也同时得到了高表达量的抗体。     

Virtual screening of a combinatorial library of enantioselective catalysts with chirality codes and counterpropagation neural networks

Conformation-independent chirality codes, radial distribution function (RDF) codes, and indicator variables are implemented to represent 1914 catalysts in a combinatorial library which was tested by Riant and co-workers for the asymmetric-hydrogen transfer to acetophenone. The catalysts which combine a metallic center with a chiral ligand have been evaluated in terms of both enantiomeric excess and yield. A counterpropagation neural network (CPG NN) was trained with a small fraction of the library to predict the performance of catalysts, and applied to the virtual screening of the remaining library. Selection of < 20.8% of the virtual library with the highest predicted performance enables to identify up to 85.5% of the best catalysts. The approach illustrates a chemoinformatic method to assist the optimization of resources for the screening of enantioselective catalysts.     

Bipartite design of a self-fibrillating protein copolymer with nanopatterned peptide display capabilities

The development of biomatrices for technological and biomedical applications employs self-assembled scaffolds built from short peptidic motifs. However, biopolymers composed of protein domains would offer more varied molecular frames to introduce finer and more complex functionalities in bioreactive scaffolds using bottom-up approaches. Yet, the rules governing the three-dimensional organization of protein architectures in nature are complex and poorly understood. As a result, the synthetic fabrication of ordered protein association into polymers poses major challenges to bioengineering. We have now fabricated a self-assembling protein nanofiber with predictable morphologies and amenable to bottom-up customization, where features supporting function and assembly are spatially segregated. The design was inspired by the cross-linking of titin filaments by telethonin in the muscle sarcomere. The resulting fiber is a two-protein system that has nanopatterned peptide display capabilities as shown by the recruitment of functionalized gold nanoparticles at regular intervals of ～ 5 nm, yielding a semiregular linear array over micrometers. This polymer promises the uncomplicated display of biologically active motifs to selectively bind and organize matter in the fine nanoscale. Further, its conceptual design has high potential for controlled plurifunctionalization.     

Immobilization of malarial (Plasmodium falciparum) dihydrofolate reductase for the selection of tight-binding inhibitors from combinatorial library

A simple procedure for selection of tight-binding inhibitors of mutant dihydrofolate reductases from Plasmodium falciparum (PfDHFRs) based on preferential binding to the enzyme immobilized on a Sepharose column has been described. PfDHFRs with a cysteine residue at the C-terminal have been prepared in order to immobilize to a thiopropyl-Sepharose gel via S-S linkage. The amount of immobilized DHFRs was estimated to be 4-5 mg/g of dried gel, and the activities of bound DHFRs were comparable to that of free enzymes. The prepared immobilized enzyme has been used for the selection of tight-binding inhibitors from combinatorial libraries, based on the affinities of each ligand with the enzyme. Free ligands were then identified and analyzed quantitatively by high-performance liquid chromatography-mass spectrometry, and the components with high binding affinity of the library could thus be realized. Results could be confirmed by quantitative analysis of the bound ligands released from the enzyme by guanidine hydrochloride treatment.     

Invention as a combinatorial process: evidence from US patents

Invention has been commonly conceptualized as a search over a space of combinatorial possibilities. Despite the existence of a rich literature, spanning a variety of disciplines, elaborating on the recombinant nature of invention, we lack a formal and quantitative characterization of the combinatorial process underpinning inventive activity. Here, we use US patent records dating from 1790 to 2010 to formally characterize invention as a combinatorial process. To do this, we treat patented inventions as carriers of technologies and avail ourselves of the elaborate system of technology codes used by the United States Patent and Trademark Office to classify the technologies responsible for an invention''s novelty. We find that the combinatorial inventive process exhibits an invariant rate of ‘exploitation’ (refinements of existing combinations of technologies) and ‘exploration’ (the development of new technological combinations). This combinatorial dynamic contrasts sharply with the creation of new technological capabilities—the building blocks to be combined—that has significantly slowed down. We also find that, notwithstanding the very reduced rate at which new technologies are introduced, the generation of novel technological combinations engenders a practically infinite space of technological configurations.     

Wavelength-dependent diffraction patterns from a liquid crystal display

Liquid crystal displays (LCDs) are invaluable for a variety of optical applications, including the encoding of programmable diffractive optical elements. The pixel structure in these devices produces a set of diffracted orders of which the central order is the strongest. In most devices that we have examined, the intensity distribution of the diffraction pattern is independent of the wavelength of the illuminating light. Recently we have been examining the performance of LCDs having very small pixel sizes. We compare results for two devices from the same manufacturer. One of them exhibits the normal behavior. For the other, we find surprisingly strong wavelength dependence. The diffraction pattern varies from having most of the energy in the zero order for long wavelengths to having the energy distributed among 50-60 orders as the wavelength decreases. We attribute this behavior to a phase structure over each pixel. We analyze this behavior using a simple two-dimensional model that qualitatively explains the phenomenon. These results can be viewed in two ways--on the positive side this behavior might lead to optical logic or fan-out applications. On the negative side, there is less intensity available in the normally used zero order.     

Benzoyl derivatization as a method to improve retention of hydrophilic peptides in tryptic peptide mapping

This study exploits the increase in chromatographic retention that accrues from benzoyl derivatization of primary amines as a tool to increase sequence coverage in tryptic peptide mapping. N-hydroxysuccinamide sulfonyl benzoate quantitatively derivatizes primary amines of peptides. Introduction of the hydrophobic benzoyl moiety into peptides increased retention of peptides during reversed-phase chromatography (RPC), particularly in the case of smaller hydrophilic peptides. Short chain (1-6 amino acids) tryptic fragments of model proteins lysozyme, myoglobin, and cytochrome c derivatized with N-hydroxysuccinamide sulfonyl benzoate eluted in the linear acetonitrile gradient. Application of benzoyl derivatization was further extended to achieve complete sequence coverage of a therapeutic protein, recombinant human growth hormone, and in detection of single amino acid polymorphism.     

Investigation of solid-phase peptide synthesis by the near-infrared multispectral imaging technique: a detection method for combinatorial chemistry.

A near-infrared (NIR) multispectral imaging spectrometer was used to monitor solid-phase peptide synthesis. This imaging spectrometer has fast scanning ability and high sensitivity because it is based on an acousto-optic tunable filter and a NIR InGaAs focal plane array camera. This NIR imaging instrument possesses all the advantages of conventional NIR spectrometers; namely, it can be used for noninvasive monitoring of the reactions and identification of the products during the solid-phase peptide synthesis of glycine, alanine, and valine mediated by aminomethylstyrene resin beads. The reaction was determined by monitoring either the decrease of the band at 1529 nm, which is due to the amine group on the beads, or the increase of the amide band generated at 1483 nm. The amine band at 1529 nm was also used to determine the presence of the Fmoc protecting groups and the efficiency of its removal. More importantly, this NIR imaging spectrometer has additional features that conventional NIR spectrometers cannot offer; namely, its ability to measure spectra at different positions within a sample. This feature was utilized for the first demonstration in which reactions of three different solid-phase peptide syntheses (in a three-compartment cell) were simultaneously monitored. As expected, the kinetics obtained for three reactions are similar to those obtained when the each of the reactions was individually determined. In this study, data recorded by 16 x 16 pixels were used to calculate a spectrum for each sample. However, a relatively good spectrum can be obtained by using data recorded by a single pixel. Since the NIR camera used in this camera is equipped with 240 x 320 pixels, this NIR mutispectral imaging technique is not limited to the three-compartment cell used in this study but rather can be used as the detection method for the solid-phase peptide synthesis in combinatorial chemistry.     

Spontaneous integration of transmembrane peptides into a bacterial magnetic particle membrane and its application to display of useful proteins

An antimicrobial peptide, temporin L, and its derivative (TL-A2) were employed as anchor peptides and displayed streptavidin on a bacterial magnetic particle (BMP) membrane. The ribotoxin L3 loop (L3) and the arginine-chain peptide (R(12)), which are carrier peptides permeable to eukaryotic cell membranes, were also used. The peptides were labeled with a fluorescent dye, 4-fluoro-7-nitrobenzofurazan (NBD), at the N-terminal region (NBD-peptides) and mixed with BMPs. A specific integration of NBD-temporin L into a BMP membrane was observed. The basic amino acids in temporin L played an important role in the integration into BMPs. Biotin conjugated to the N-terminus of temporin L was integrated into a BMP membrane. The C-terminus of temporin L was incorporated into a BMP membrane, and the N-terminus was located on the BMP membrane surface. The present study shows that temporin L is a stable molecular anchor on BMPs by the binding of soluble protein to the N-terminus.     

Fluorescence-based peptide labeling and fractionation strategies for analysis of cysteine-containing peptides

This study demonstrates that 1,5-I-AEDANS (5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid) can be used as a versatile fluorescence-based peptide quantification tool and provides readily interpretable tandem mass spectra for de novo peptide sequencing. Two AEDANS-cysteinyl-peptide fractionation strategies were evaluated. One AEDANS-cysteinyl-peptide fractionation strategy employs immobilized metal affinity chromatography (IMAC) to recover AEDANS-labeled peptides and reduce the complexity of peptide mixtures. In an alternate solid-phase approach, 1,5-I-AEDANS was coupled to an o-nitrobenzyl-based photocleavable resin to produce a resin that can label and isolate thiols and cysteine-containing peptides with a modified-AEDANS label (mAEDANS: 5-((4-amino-4-oxobutanoyl){2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid). This fractionation protocol enriches cysteine-containing peptides more specifically than the IMAC strategy. Using micro-LC-ESI-MS with an on-line fluorescence detector and a Q-TOF mass spectrometer, we generated fluorescence-based elution profiles and corresponding positive ion mass spectra of AEDANS-labeled peptides. This study demonstrates that AEDANS-peptides produce positive ion ESI-MS mass spectra with detection limits comparable to those of the unlabeled peptide. Collision-induced dissociation (CID) of fluorescent AEDANS-peptides revealed readily interpretable product ion spectra with the label intact. Similar to the AEDANS-labeled peptide, an mAEDANS-labeled thiol is fluorescent and CID of a mAEDANS-labeled peptide also reveals an interpretable product ion spectrum with the label intact.     

Nonretentive solid-phase extraction of phosphorylated peptides from complex peptide mixtures for detection by matrix-assisted laser desorption/ionization mass spectrometry

Widespread interest in protein phosphorylation has led to the development of a variety of methods for the analysis of phosphoproteomes of different types of organisms. Many applications involve pretreatment of the sample before mass spectrometric measurement and can crucially improve the detection efficiency of individual phosphopeptides. Despite intense research efforts, separation and extraction of phosphorylated peptides, especially multiphosphorylated ones, remain challenging tasks and need to be further explored and expanded with unconventional approaches. In this study, we describe the application of nonretentive solid-phase extraction (SPE) to the analysis of phosphopeptides using the highly cross-linked polystyrene-divinylbenzene material Strata-X. This study indicates that the procedure allows for the preferential extraction of phosphopeptides regardless of their extent of phosphorylation. The Strata-X material primarily retains nonphosphorylated peptides by hydrophobic interaction, whereas the inherent hydrophilicity of phosphorylated peptides leads to their partitioning into the aqueous phase. Phosphopeptides that were rapidly segregated out of tryptic digest mixtures and collected in the early aqueous fractions generated intense signals in mass spectra. The method was developed using SPE Strata-X columns, then suited for detection and sequencing of phosphopeptides by miniaturizing the system to the scale of custom-made microcolumns. This provided fast isolation of phosphopeptides from protein digests along with direct MALDI on-target deposition. The possibility of on-target washing during sample preparation is also presented.     

Three-dimensional display of a horizontal-parallax-only hologram

We propose a three-dimensional (3D) holographic display by converting an optically recorded complex full-parallax (FP) hologram to an off-axis horizontal-parallax-only (HPO) hologram. First, we record the complex FP hologram of an object using optical scanning holography. We then convert the complex FP hologram to an off-axis HPO hologram through fringe-matched Gaussian low-pass filtering and with the introduction of an off-axis reference. Finally, we reconstruct the off-axis HPO hologram optically using an amplitude-only spatial light modulator. Until now, only computer-generated HPO holograms have been displayed optically. To the best of our knowledge, this is the first demonstration of a 3D display of an optically recorded HPO hologram.