Homogeneous enzyme-linked binding assay for studying the interaction of lectins with carbohydrates and glycoproteins

A simple and rapid homogeneous enzyme-linked binding assay method for studying lectin-carbohydrate interactions is described. The method is based on the homogeneous inhibition of appropriate enzyme-saccharide conjugates by specific carbohydrate-binding lectins. In the presence of carbohydrate structures recognized by the lectins, enzyme activity is regained in an amount of proportional to the concentration of carbohydrate. The new method can be used to rapidly assess the relative carbohydrate specificity of the various lectins and for the selective analytical detection of simple saccharides and complex glycoproteins. Indeed, when Jacalin lectin is used in conjunction with a malate dehydrogenase-galactose conjugate, selective measurement of human IgA (immunoglobulin A) at microgram per milliliter levels in less than 10 min is possible. The potential for using this analytical methodology for determining changes in the carbohydrate structure of intact recombinant glycoproteins is also discussed.     

Screening of combinatorial libraries for substrate preference by mass spectrometry

We present a rapid screening method for monitoring enzyme specificity using both combinatorial chemistry and mass spectrometry where, as an example, the substrate specificity of peptidylglycine alpha-amidating enzyme was determined and compared against a conventional quantitative technique. Whereas alternative methods for library screening are generally limited to certain enzymes and can present difficulties in the synthesis or derivatization of potential substrates, the approach we call chirality-based isotope labeling for a library of substrates (CHILLS) does not fall short to such limitations, since we exploit the inherent stereospecificity of enzymes to determine preferred substrates. Additionally, the CHILLS method generates accurate results, as compared to typical screening procedures that require tedious method development, because the synthesized library contains a structurally similar internal standard for each individual library component in order to quantitate the progress of enzymatic reactions.     

High-throughput screening for enzyme inhibitors using frontal affinity chromatography with liquid chromatography and mass spectrometry

This work presents new frontal affinity chromatography (FAC) methodologies for high-throughput screening of compound libraries, designed to increase screening rates and improve sensitivity and ruggedness in performance. A FAC column constructed around the enzyme N-acetylglucosaminyltransferase V (GnT-V) was implemented in the identification of potential enzyme inhibitors from two libraries of trisaccharides. Effluent from the FAC column was fractionated, sequentially processed via LC/MS, and referenced to a similar analysis through a control FAC column lacking the enzyme. The resulting multidimensional data sets were compared across corresponding sample and control fractions to identify binders, in a semiautomated approach. A strong binder in the protonated form at m/z 795 was identified from the first library of 81 compounds, exhibiting an estimated Kd value of 0.3 microM. Other binders yielded Kd values ranging from 0.35 to 3.35 microM. To demonstrate the improvement in performance of this FAC-LC/MS approach over the conventional online FAC/MS approach, 15 compounds from this library were blended with a second library of 1000 synthetic trisaccharides and screened against GnT-V. All ligands in the 15-compound set were identified in this larger screen, and no ligands of greater affinity than compound 1 were found. Our results show that FAC-LC/MS is a reliable method for screening large compound libraries directly and useful for large-scale ligand discovery initiatives.     

Method for screening and MALDI-TOF MS sequencing of encoded combinatorial libraries

We describe a new method for encoded synthesis, efficient on-resin screening, and rapid unambiguous sequencing of combinatorial peptide libraries. An improved binary tag system for encoding peptide libraries during synthesis was designed to facilitate unequivocal assignment of isobaric residues by MALDI-TOF MS analysis. The improved method for encoded library synthesis was combined with a new versatile on-resin screening strategy that permitted multiple stages and types of screening to be employed successively on one library under mild conditions. The new method facilitated a combinatorial study of transglutaminase (TGase) enzyme substrate peptides, revealing new details of the effect of amino acid composition on TGase substrates. The approach was first demonstrated for an encoded library (130,321 compounds) of lysine pentapeptide substrates of TGase, synthesized using the "split-mix" method. The library was reacted on-resin with TGase enzyme and a soluble desthiobiotin labeled glutamine substrate. Initial screening was performed by adsorbing streptavidin-coated magnetic microparticles onto library beads, followed by magnetic separation. The differential binding affinities of desthiobiotin and biotin for streptavidin were exploited to release the magnetic microparticles and regenerate the desthiobiotin-labeled resin beads for further screening by flow-cytometry-based automated bead sorting, resulting in 345 beads that were sequenced by MALDI-TOF MS analysis. A second library consisted of encoded glutamine hexapeptide substrates, which was reacted on-resin with TGase enzyme and a soluble desthiobiotin-labeled cadaverine. Two-stage screening identified 267 glutamine peptides as TGase-reactive, of which 21 were further analyzed by solution-phase enzyme kinetics. Kinetic results indicated that the peptide PQQQYV from the library has a 68-fold greater substrate specificity than the best known glutamine substrate QQIV. The new encoding and screening strategies described here are expected to be broadly applicable to synthesis and screening of combinatorial peptide libraries in the future.     

Accelerated exploration of TRIP metallic glass composite by laser additive manufacturing

Introducing transformation-induced plasticity(TRIP)effect into bulk metallic glass composites(BMGCs)is an effective route to improve their ductility and strain-hardening ability.Since the morphology and structure of the crystalline austenite phases responsible for the TRIP phenomenon are strongly depen-dent on the alloy composition and cooling rate during freezing,distinguishing the optimal cases from a vast variety of candidates is the primary task of exploring TRIP BMGCs.However,without a suitable theoretical guidance,the exploration of BMGCs is usually performed via the traditional trial-and-error route,making the BMGC development extremely time consuming and labor intensive.Here,we present a novel high-throughput strategy to accelerate the exploration process of TRIP BMGCs.The efficiency of this strategy was demonstrated on a well-studied Cu-Zr-Al alloy system.A screening library,comprised by 121 cylindrical samples with different conditions,was rapidly prepared by laser additive manufacturing(LAM).The phases of the library were efficiently identified by micro-area X-ray diffraction(M-XRD)to screen the optimal compositions and cooling rates that precipitate only B2-CuZr phase.The distribution uniformity of the B2-CuZr phase was further evaluated based on digital image processing technology to screen the candidates of better ductility.The high-throughput results are in good agreement with the pre-vious casting investigations of discrete samples,confirming the validity of the present high-throughput strategy.     

Congruent strategies for carbohydrate sequencing. 1. Mining structural details by MSn

This report is the first in a series of three focused on establishing congruent strategies for carbohydrate sequencing. The reports are divided into (i) analytical considerations that account for all aspects of small oligomer structure by MSn disassembly, (ii) database support using an ion fragment library and associated tools for high-throughput analysis, and (iii) a concluding algorithm for defining oligosaccharide topology from MSn disassembly pathways. The analytical contribution of this first report explores the limits of structural detail exposed by ion trap mass spectrometry with samples prepared as methyl derivatives and analyzed as metal ion adducts. This data mining effort focuses on correlating the fragments of small oligomers to stereospecific glycan structures, an outcome attributed to a combination of metal ion adduction and analyte conformation. Facile glycosidic cleavage introduces a point of lability (pyranosyl-1-ene) that upon collisional activation initiates subsequent ring fragmentation. Product masses and ion intensities vary with interresidue linkage, branching position, and monomer stereochemistry. Excessive fragmentation is the property of small oligomers where collisional energy within a smaller number of oscillators dissipates through extensive fragmentation. The procedures discussed in this report are unified into a singular strategy using an ion trap mass spectrometer with the sensitivity expected for electron multiplier detection. Although a small set of structures have been discussed, the basic principles considered are fully congruent, with ample opportunities for expansion.     

Charge heterogeneity of monoclonal antibodies by multiplexed imaged capillary isoelectric focusing immunoassay with chemiluminescence detection

Characterization of charge heterogeneity of recombinant monoclonal antibodies (mAbs) requires high throughput analytical methods to support clone selection and formulation screens. We applied the NanoPro technology to rapidly measure relative charge distribution of mAbs in early stage process development. The NanoPro is a multiplexed capillary-based isoelectric immunoassay with whole-column imaging detection. This assay offers specificity, speed and sensitivity advantages over conventional capillary isoelectric focusing (CIEF) platforms. After CIEF, charge variants are photochemically immobilized to the wall of a short coated capillary. Once immobilized, mAbs are probed using a secondary anti-IgG conjugated with horseradish peroxidase. After flushing away excess reagents, secondary antibodies bound to their targets are then detected by chemiluminescence upon incubation with peroxidase reactive substrates. Charge heterogeneity as determined by chemiluminescence was similar to that measured by conventional CIEF technology with absorbance detection for purified mAbs and contaminated mAbs derived directly from host cellular extract. Upon method optimization, the automated CIEF immunoassay was applied to several mAbs of varying isoelectric points, demonstrating the suitability of NanoPro as a rugged high-throughput product characterization tool. Furthermore, qualification of detection sensitivity, precision, and dynamic range are reported with discussion of its advantages as an alternative approach to rapidly characterize charge variants during process development of mAbs.     

High-throughput method for the production and analysis of large natural product libraries for drug discovery

High-throughput methods were applied to the production, analysis, and characterization of libraries of natural products in order to accelerate the drug discovery process for high-throughput screening in the pharmaceutical and biotechnology industries. Library production integrates automated flash chromatography, solid-phase extraction, filtration, and high-throughput parallel four-channel preparative high-performance liquid chromatography to obtain the libraries in 96- or 384-well plates. Libraries consist of purified fractions with approximately one to five compounds per well. Libraries are analyzed prior to biological screening by a high-throughput parallel eight-channel liquid chromatography-evaporative light scattering detection-mass spectrometry system to determine the molecular weight, number, and quantity of compounds in a fraction. After biological screening, active fractions are rapidly purified at the microgram level and individual compounds are rescreened for confirmation of activity. Structures of active compounds are elucidated by NMR spectroscopy and mass spectrometry. Utilization of a novel microcoil probe allows NMR data to be gathered on 50 microg. As a demonstration, a library was made from the stem bark of Taxus brevifolia. Biological screening in the National Cancer Institute's in vitro panel of three cancer cell lines demonstrates that the process enables the discovery of active anticancer compounds not detected in the flash fractions from which the library originates.     

High-Throughput Multi-Plume Pulsed-Laser Deposition for Materials Exploration and Optimization

A high-throughput multi-plume pulsed-laser deposition(MPPLD) system has been demonstrated and compared to previous techniques. Whereas most com binatorial pulsedlaser deposition(PLD) systems have focused on achieving thickness uniformity using sequential multilayer deposition and masking followed by post-deposition annealing, MPPLD directly deposits a compositionally varied library of compounds using the directionality of PLD plumes and the resulting spatial variations of deposition rate. This system is more suitable for high-throughput compound thin-fllm fabrication.     

Biomaterials functionalization using a novel peptide that selectively binds to a conducting polymer

The goal in biomaterial surface modification is to retain a material's bulk properties while modifying only its surface to possess desired recognition and specificity. Here we develop a unique strategy for surface functionalization of an electrically conductive polymer, chlorine-doped polypyrrole (PPyCl), which has been widely researched for various electronic and biomedical applications. An M13 bacteriophage library was used to screen 10(9) different 12-mer peptide inserts against PPyCl. A binding phage (phiT59) was isolated, and its binding stability and specificity to PPyCl was assessed using fluorescence microscopy and titer count analysis. The relative binding strength and mechanism of the corresponding 12-mer peptide and its variants was studied using atomic force microscopy and fluorescamine assays. Further, the T59 peptide was joined to a cell adhesive sequence and used to promote cell attachment on PPyCl. This strategy can be extended to immobilize a variety of molecules to PPyCl for numerous applications. In addition, phage display can be applied to other polymers to develop bioactive materials without altering their bulk properties.     

Simulation of evolution-selected propeptide by high-throughput selection of a peptidomimetic inhibitor on a capillary DNA sequencer platform

Many proteinases, including gelatinase B/MMP-9, fulfill crucial regulatory or effector functions in disease states and may be pharmacologically targeted by specific inhibitors. Denatured collagen type II provides one of the best gelatinase B substrates, and the characteristics of its cleavage were employed to define the requirements of a novel optimal substrate probe. A synthetic fluorescent derivative was used for the development of a new high-throughput technology for the selection of inhibitors on the principles of sensitivity of confocal fluorescence detection, resolution capacity of capillary electrophoresis, and multichannel power of DNA sequencers. Combinatorial chemical synthesis of a library of peptide-based inhibitors, library deconvolution, high-throughput screening, isolation, and mass spectrometric techniques enabled us to identify a novel single-peptide gelatinase B inhibitor. A notable finding is that the in vitro-selected inhibitor mimics many of the characteristics of the evolution-selected MMP propeptide sequence.     

Packed column supercritical fluid chromatography/mass spectrometry for high-throughput analysis

A supercritical fluid chromatograph was interfaced to a mass spectrometer, and the system was evaluated for applications requiring high sample throughput. Experiments presented demonstrate the high-speed separation capability of supercritical fluid chromatography (SFC) and the effectiveness of supercritical fluid chromatography/mass spectrometry (SFC/MS) for fast, accurate determinations of multicomponent mixtures. A high-throughput liquid chromatography/mass spectrometry (LC/MS) analysis cycle time is reduced 3-fold using our general SFC/MS high-throughput method, resulting in substantial time saving for large numbers of samples. Unknown mixture characterization is improved due to the increased selectivity of SFC/MS compared to LC/MS. This was demonstrated with sample mixtures from a 96-well combinatorial library plate. In this paper, we report a negative mode atmospheric pressure chemical ionization (APCI) method for SFC/MS suitable for most of the components in library production mixtures. Flow injection analysis (FIA) also benefits from this SFC/MS system. A broader range of solvents is amenable to the SFC mobile phase compared with standard LC/MS solvents, and solutes elute more rapidly from the SFC/MS system, reducing sample carryover and cycle time. Finally, our instrumental setup allows for facile conversion between LC/MS and SFC/MS modes of operation.     

Automated high-throughput liquid-liquid extraction for initial purification of combinatorial libraries

An automated high-throughput liquid-liquid extraction (LLE) methodology has been developed and utilized for the initial purification of the combinatorial library samples containing unreacted amines and other water-soluble byproducts or impurities. Various extraction solvents were evaluated along with different extraction devices. The LLE method was automated using 96-well-format plates and a robotic liquid-handling workstation. In the optimized LLE method, crude combinatorial library samples were dissolved in a water-immiscible organic solvent, butyl acetate, and added to each well in a 96-well-format plate packed with an inert support material coated with hydrochloric acid. Separation occurs based on the partitioning of the compounds between two liquid phases. Product recovery, purity, and amine removal efficiency were determined by HPLC with and without precolumn derivatization. The automated method was successfully applied to the cleanup of some representative combinatorial library samples with greater than 98% amine removal and an average product purity of 90%. The application of the automated high-throughput LLE method should greatly reduce the labor, time, and cost associated with the purification of combinatorial libraries.     

TOF-SIMS analysis of a 576 micropatterned copolymer array to reveal surface moieties that control wettability

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used in a high-throughput fashion to obtain mass spectra from the surfaces of 576 novel acrylate-based polymers, synthesized using a combinatorial approach and in a micropatterned format. To identify variations in surface chemistry within the library, principal component analysis (PCA) was used. PCA clearly identified surface chemical commonality and differences within the library. The TOF-SIMS spectra were also used to determine the relationship between water contact angle (WCA) and the surface chemistry of the polymer library using partial least-squares regression (PLS). A good correlation between the TOF-SIMS data from the novel polymers and water contact angle was obtained. Examination of the PLS regression vector allowed surface moieties that correlate with high and low WCA to be identified. This in turn provided an insight into molecular structures that significantly influence wettability. This study demonstrates that multivariate analysis can be successfully applied to TOF-SIMS data from a large library of samples and highlights the potential of these techniques for building complex surface property/chemistry models.     

Microrheology as a tool for high-throughput screening

Microrheology can be used for high-throughput screening of the rheological properties of sample libraries of complex fluids. Two passive techniques are particularly suitable: video microscopy and diffusing-wave spectroscopy. The techniques complement each other very well and can be applied to samples that offer different experimental challenges. We offer a thorough analysis of the strengths and limitations of microrheology with the emphasis on high-throughput applications. To illustrate the potential of microrheology, results are presented for two representative cases: the rheological screening of aqueous solutions of a block copolypeptide library and the rheological phase diagram of a water/surfactant/salt system.     

High-throughput analysis of natural product compound libraries by parallel LC-MS evaporative light scattering detection

The application of an 8-way fully automated parallel LC-MS-ELSD system to the analysis of a library of 96 structurally diverse natural products is described. A 10-min separation incorporating a universal gradient allowed elution of 86 of these 96 compounds, all of which were detected by positive or negative mode electrospray ionization in conjunction with ELSD. This method is demonstrated to be one of the most universal means of detection for polar, nonvolatile, thermally labile natural products. It also allows an 8-fold increase in throughput. The analysis and profiling of constituents present in a library derived from plant material of Sarcostemma hirtellum, in terms of their retention times and mass spectra, are shown. This rapid characterization of plant constituents in terms of compound libraries is important in searching for new biologically active compounds. As a high-throughput tool to support our natural product discovery program, this method has been successfully used to analyze a library of 36,000 partially purified fractions derived from plant materials.     

可用于快速高温热处理的多束激光并行加热系统

快速、高通量的实验方法对材料基因工程技术的发展具有重要意义。高温热处理对无机非金属材料的制备往往是必不可少的环节, 然而针对材料基因工程所需的材料样品库快速热处理技术目前尚是空白。本文报道了一种可用于快速高温热处理阵列样品的多束激光并行加热系统, 介绍了多束激光并行加热系统的设计原理、工作方式、结构细节和软件设计。该装置具有激光加热时间、功率、光斑大小可独立调节、自动化程度高等优点, 最大单束激光功率大于100 W, 每束激光的最大稳定加热温度约为2000 ℃。作为典型的应用示范, 进行了一系列Ce³⁺掺杂钇铝石榴石发光陶瓷的烧结实验,加热温度分别为1400、1500和1600 ℃, 保温时间分别为180、360和540 s, 仪器按照设置好的加热位置和加热曲线自动运行。结果表明: 采用多束激光并行加热系统, 可以在几分钟的热处理时间内获得结晶度和发光性能良好的烧结样品, 比传统制备工艺十余小时的烧结时间大幅缩短。这将为高温热处理的条件筛选和高通量制备提供一种节能省时的新技术方案。     

Enzyme reactions in nanoporous, picoliter volume containers

Advancements in nanoscale fabrication allow creation of small-volume reaction containers that can facilitate the screening and characterization of enzymes. A porous, ～19 pL volume vessel has been used in this work to carry out enzyme reactions under varying substrate concentrations. Assessment of small-molecule and green fluorescent protein diffusion from the vessels indicates that pore sizes on the order of 10 nm can be obtained, allowing capture of proteins and diffusive exchange of small molecules. Glucose oxidase and horseradish peroxidase can be contained in these structures and diffusively fed with a solution containing glucose and the fluorogenic substrate amplex red through the engineered nanoscale pore structure. Fluorescent microscopy was used to monitor the reaction, which was carried out under microfluidic control. Kinetic characteristics of the enzyme (K(m) and V(max)) were evaluated and compared with results from conventional scale reactions. These picoliter, nanoporous containers can facilitate quick determination of enzyme kinetics in microfluidic systems without the requirement of surface tethering and can be used for applications in drug discovery, clinical diagnostics, and high-throughput screening.     

Research fronts in library and information science in Spain (1985–1994)

Publications and author cocitations in library and information science in Spain during the period from 1985 to 1994 were analyzed as a measure of the structure, specificity and composition of research fronts in this country. A cocitation matrix developed from an ad hoc database was subjected to cluster analysis, multidimensional scaling and principal components analysis. The resulting cocitation maps identified specific areas of research and their knowledge bases. We inferred the degree of consolidation of the discipline of library and information science, and of the subdisciplines informetrics, librarianship and university affiliation, from the research activities revealed. In this respect, the conclusions from the study show the existence of several research fronts in Spanish literature the contents of which are in most cases difficult to compare with those in other countries. A lesser degree of maturity of research in this field is shown.