Microarray: a versatile platform for high-throughput functional proteomics

The advent of microarray technologies has dramatically accelerated the functional study of proteins, including enzymes (catalomics) in a proteome. Herein, we review recent advances and exciting new developments of microarrays in high-throughput functional proteomics.     

Biochemical reactions in metabolite-protein interaction

An update on the recently developed chemical proteomics called activity-based protein profiling (ABPP) has been reviewed. ABPP is able to identify proteins interacted either covalently or non-covalently with metabolites significantly, which will facilitate the characterization of specific metabolite regulating proteins in human disease progression.     

Proteomics on a chip: promising developments

The field of proteomics is expanding rapidly due to the completion of the human genome and the realization that genomic information is often insufficient to comprehend cellular mechanisms. This considerable expansion of proteomics towards high-throughput platforms is stressing its current technical capabilities. In recent years, technologies in microfluidic and array technologies have appeared for proteomics. These novel approaches might help solve current technical challenges in proteomics. This review presents a general survey of the recent development in microfluidic and array technologies from a proteomics perspective.     

Enzymatic determination of fenitrothion by cholinesterase and acetylcholinesterase on fluorogenic substrates

 The action of the enzymes acetylcholinesterase and cholinesterase on the substrates indoxylacetate and 2-naphthyl acetate was studied kinetically. Both enzymes convert the substrates to highly fluorescent products (3-hydroxy-indole and 2-naphthol, respectively). The kinetic curves present the initial rate as the variation of fluorescence for a unity of time (ΔF/Δt) against the substrate concentration. This enzymatic reaction was investigated in presence of the inhibitor organophosphate pesticide fenitrothion. From the kinetic curves the enzymatic parameters and enzyme and substrate concentrations used for the calibration curve can be obtained. Four simple spectrofluorimetric methods for the enzymatic determination of fenitrothion were developed showing detection limits between 5.5 to 19.5 μmol/l, depending on the enzyme and the substrate used. The precisions (R.S.D.) of the methods were between 1.6 and 9.6%. A comparative study of the kinetic enzymatic parameters and the detection limits was performed. A good correlation was obtained between inhibition constants and the detection limit. Received: 21 February 1996/Revised: 2 July 1996/Accepted: 9 July 1996     

Carbohydrate microarrays: an advanced technology for functional studies of glycans

The biological significance of glycans in the post-genomic era requires the development of new technologies to enable functional studies of carbohydrates in a high-throughput manner. Recently, carbohydrate microarrays have been exploited as an advanced technology for this purpose. Efficient immobilization methods for carbohydrate probes on the proper surface are essential for the successful fabrication of carbohydrate microarrays. Up to date, several techniques have been developed to attach simple or complex carbohydrates to a solid surface. The developed glycan microarrays have been applied for functional glycomics, drug discovery, and diagnosis. In this concept article, we discuss the progress of immobilization methods of carbohydrates on solid surfaces, their potential uses for biological research and biomedical applications, and possible solutions for some remaining challenges to improve this new technology.     

High performance protein microarrays based on glycidyl methacrylate-modified polyethylene terephthalate plastic substrate

There is a great challenge to immobilize high density of probe molecules for high performance protein microarrays, and this is achieved in this work by using polyethylene terephthalate (PET) plastic substrate onto which glycidyl methacrylate (GMA) photopolymer is grafted under mild conditions to introduce high density of epoxy groups for covalent immobilization of proteins. The poly(GMA)-grafted PET (PGMA-PET) surface was characterized with atomic force microscope (AFM) and attenuated total reflectance Fourier transform infra-red (ATR-FTIR) spectroscopy. For high density of protein immobilization and good quality of microspots, experiments were conducted to optimize the printing buffer, and an optimal buffer was found out to be PBS with 10% glycerol + 0.003% triton X-100. According to the studies of loading capacity and immobilization kinetics, the optimal protein probe concentration and incubation time for the efficient immobilization are 200 μg mL⁻¹ and 8 h, respectively. The performance of the PGMA-PET-based protein microarrays is evaluated with sandwich immunoassay using rat IgG and anti-rat IgG as model proteins, demonstrating a limit of detection (LOD) of 10 pg mL⁻¹ and a dynamic range of five orders of magnitude which are better than or very comparable with the reported or commercially available immunoassays, while providing a high-throughput approach. The work renders a simple and economic method to manufacture high performance protein microarrays and is expected to have great potentials in broad applications related to clinic diagnosis, drug discovery and proteomic research.     

糖芯片的检测及应用

糖芯片技术具有样品少、通量高和特异性强等优点,是一种糖组学研究的新的技术平台和强大的分析工具,已经广泛用于糖和蛋白质的特异性作用、酶活性和抑制剂、病毒入侵机理、细菌检测和免疫反应等方面的研究.本文简要介绍了糖芯片的原理、制备和信号的检测技术(荧光标记法、质谱法、SPR法等),分析了糖芯片在各个领域的应用及其发展前景.     

High-Throughput Proteomics Enabled by a Photocleavable Surfactant

Mass spectrometry (MS)-based proteomics provides unprecedented opportunities for understanding the structure and function of proteins in complex biological systems; however, protein solubility and sample preparation before MS remain a bottleneck preventing high-throughput proteomics. Herein, we report a high-throughput bottom-up proteomic method enabled by a newly developed MS-compatible photocleavable surfactant, 4-hexylphenylazosulfonate (Azo) that facilitates robust protein extraction, rapid enzymatic digestion (30 min compared to overnight), and subsequent MS-analysis following UV degradation. Moreover, we developed an Azo-aided bottom-up method for analysis of integral membrane proteins, which are key drug targets and are generally underrepresented in global proteomic studies. Furthermore, we demonstrated the ability of Azo to serve as an “all-in-one” MS-compatible surfactant for both top-down and bottom-up proteomics, with streamlined workflows for high-throughput proteomics amenable to clinical applications.     

Microarray technology as a universal tool for high-throughput analysis of biological systems

Over the last years microarray technology has become one of the principal platform technologies for the high-throughput analysis of biological systems. Starting with the construction of first DNA microarrays in the 1990s, microarray technology has flourished in the last years and many different new formats have been developed. Peptide and protein microarrays are now applied for the elucidation of interaction partners, modification sites and enzyme substrates. Antibody microarrays are envisaged to be of high importance for the high-throughput determination of protein abundances in translational profiling approaches. First cell microarrays have been constructed to transform microarray technology from an in vitro technology to an in vivo functional analysis tool. All of these approaches share a common prerequisite: the solid support on which they are generated. The demands on this solid support are thereby as manifold as the applications themselves. This review is aimed to display the recent developments in surface chemistry and derivatization, and to summarize the latest developments in the different application areas of microarray technology.     

Novel molecular tumour classification using MALDI–mass spectrometry imaging of tissue micro-array

The development of tissue micro-array (TMA) technologies provides insights into high-throughput analysis of proteomics patterns from a large number of archived tumour samples. In the work reported here, matrix-assisted laser desorption/ionisation–ion mobility separation–mass spectrometry (MALDI–IMS–MS) profiling and imaging methodology has been used to visualise the distribution of several peptides and identify them directly from TMA sections after on-tissue tryptic digestion. A novel approach that combines MALDI–IMS–MSI and principal component analysis–discriminant analysis (PCA–DA) is described, which has the aim of generating tumour classification models based on protein profile patterns. The molecular classification models obtained by PCA–DA have been validated by applying the same statistical analysis to other tissue cores and patient samples. The ability to correlate proteomic information obtained from samples with known and/or unknown clinical outcome by statistical analysis is of great importance, since it may lead to a better understanding of tumour progression and aggressiveness and hence improve diagnosis, prognosis as well as therapeutic treatments. The selectivity, robustness and current limitations of the methodology are discussed.     

Probing substrate binding to Metallo-β-Lactamase L1 from Stenotrophomonas maltophilia by using site-directed mutagenesis

Background  

The metallo-β-lactamases are Zn(II)-containing enzymes that hydrolyze the β-lactam bond in penicillins, cephalosporins, and carbapenems and are involved in bacterial antibiotic resistance. There are at least 20 distinct organisms that produce a metallo-β-lactamase, and these enzymes have been extensively studied using X-ray crystallographic, computational, kinetic, and inhibition studies; however, much is still unknown about how substrates bind and the catalytic mechanism. In an effort to probe substrate binding to metallo-β-lactamase L1 from Stenotrophomonas maltophilia, nine site-directed mutants of L1 were prepared and characterized using metal analyses, CD spectroscopy, and pre-steady state and steady state kinetics.     

A multiplexed screening method for agonists and antagonists of the estrogen receptor protein

The estrogen receptor (ER) is regarded as a significant drug target because of its important physical and pathological function. In this article, we describe a novel screening method to obtain agonists and antagonists of ER. ER was immobilized onto an aldehyde-modified glass slide. The affinity of Cy3-labeled estradiol for ER protein microarrays was then determined. Two libraries, one containing 29 synthetic compounds and the other with 384 natural products that served as a model, were screened to find new ligands for ER. The IC₅₀ values obtained for tamoxifen and raloxifene were consistent with those found in the literature (4.85 × 10⁻⁷ M versus 1.74~4.23 × 10⁻⁷ M and 7.58 × 10⁻⁸ M versus 0.89~5.84 × 10⁻⁸ M, respectively). Finally, 65 active ligands (5 synthetic compounds and 60 natural products) of ER were identified. This novel method gave identical results to a conventional fluorescence polarization assay, thus verifying the accuracy of this simultaneous multireceptor screening method based on protein microarrays. The presented method is sensitive, accurate, and reliable, and shows great potential for use in high-throughput drug-screening research.     

Design and synthesis of an affinity probe that targets caspases in proteomic experiments

The field of proteomics aims to study all proteins in the human proteome. This huge task may be accelerated by using active-site directed probes which profile proteins in an activity-dependent manner. Herein, we have developed a fluorescently-labeled affinity probe containing chemical reactivity specific towards caspases. Preliminary assays and proof-of-concept experiments demonstrated that this probe exhibits strong chemical reactivity towards caspase-1 over other enzymes, capable of covalently labeling caspapse-1 over other non-caspase enzymes. This thus demonstrates its selectivity and potential in high-throughput screenings of other unknown caspases in a large-scale proteomics experiment.     

Automated filter paper assay for determination of cellulase activity

Recent developments in molecular breeding and directed evolution have promised great developments in industrial enzymes as demonstrated by exponential improvements in β-lactamase and green fluorescent protein (GFP). Detection of and screening for improved enzymes are relatively easy if the target enzyme is expressible in a suitable high-throughput screening host and a clearly defined and usable screen or selection is available, as with GFP and β-lactamase. Fungal cellulases, however, are difficult to measure and have limited expressibility in heterologous hosts. Furthermore, traditional cellulase assays are tedious and time-consuming. Multiple enzyme components, an insoluble substrate, and generally slow reaction rates have plagued cellulase researchers interested in creating cellulase mixtures with increased activities and/or enhanced biochemical properties. Although the International Union of Pure and Applied Chemists standard measure of cellulase activity, the filter paper assay (FPA), can be reproduced in most laboratories with some effort, this method has long been recognized for its complexity and susceptibility to operator error. Our current automated FPA method is based on a Cyberlabs C400 robotics deck equipped with customized incubation, reagent storage, and plate-reading capabilities that allow rapid evaluation of cellulases acting on cellulose and has a maximum throughput of 84 enzyme samples per day when performing the automated FPA.     

Prolonged durability of electroporation microarrays as a result of addition of saccharides to nucleic acids

The electroporation microarray is a useful tool for high-throughput analysis of gene functions. However, transfection efficiency is greatly impaired by storage of the microarrays, due to water evaporation from arrayed nucleotides. In this study, we aimed at evaluating the effect of saccharides and sugar alcohols, added to the solution of the plasmid DNA or small interfering RNA (siRNA). Microarrays loaded with plasmids and siRNAs were prepared with various polyols including sugars and sugar alcohols. After storage of these microarrays at different temperatures for various time periods, transfection efficiency was evaluated using human embryonic kidney cells. In the case of plasmid-loaded microarrays, addition of monosaccharides (glucose, fructose), disaccharides (trehalose, sucrose), and trisaccharide (raffinose) served to retain transfection efficiency at a reasonably high level after storage at −20 °C. The observed effects may be because moisture retention serves to maintain the solubility of DNA. In contrast, polysaccharide (dextran) and sugar alcohol (glycerol) had insignificant effects on retention of transfection efficiency. On the other hand, addition of saccharides and sugar alcohols had insignificant effects on the transfection of siRNA after storage of a microarray at 25 °C for 7 days, presumably due to the intrinsically-high solubility of siRNA which consists of short nucleotides. Figure (PPTX 3.80 MB)

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Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A real-time,fluorescence-based assay for Rho-associated protein kinase activity

Inhibitors of Rho-associated protein kinase (ROCK) enzymatic activity have been shown to reduce the invasive phenotype observed in metastatic hepatocellular carcinoma (HCC). We describe the design, synthesis, and evaluation of a direct probe for ROCK activity utilizing a phosphorylation-sensitive sulfonamido-oxine fluorophore, termed Sox. The Sox fluorophore undergoes an increase in fluorescence upon phosphorylation of a proximal amino acid via chelation-enhanced fluorescence (CHEF, ex. = 360 nm and em. = 485 nm), allowing for the direct visualization of the rate of phosphate addition to a peptide substrate over time. Our optimal probe design, ROCK-S1, is capable of sensitively reporting ROCK activity with a limit of detection of 10 pM and a high degree of reproducibility (Z’-factor = 0.6 at 100 pM ROCK2). As a proof-of-principle for high-throughput screening (HTS) we demonstrate the ability to rapidly assess the efficacy of a 78 member, small molecule library against ROCK2 using a robotics platform. We identify two previously unreported ROCK2 inhibitor scaffolds, PHA665752 and IKK16, with IC₅₀ values of 3.6 μM and 247 nM respectively. Lastly, we define conditions for selectively monitoring ROCK activity in the presence of potential off-target enzymes (PKCα, PKA, and PAK) with similar substrate specificities.     

Development of a Protein Chip to Measure PKCβ Activity

Phosphorylation of proteins by kinases plays an important role in regulating cellular processes including melanin production in the skin cells. Protein kinase C β (PKCβ) is known to be involved in phosphorylating tyrosinase, the key enzyme of melanin production, regulating the skin pigmentation process. In melanogenesis, PKCβ activates the tyrosinase by phosphorylation of its two serine residues. In this study, phosphorylation activity by PKCβ was monitored on a protein chip for the screening of depigmenting agents. As a tyrosinase mimic, 11 or 30 amino acids of the C-terminal of tyrosinase was fused with maltose-binding protein (MBP). After immobilizing the MBP-fused PKCβ substrate peptide on epoxy-treated slide surface, PKCβ reaction mix was applied over the immobilized MBP-fused PKCβ substrate peptide. Phosphorylation was detected with anti-phosphoSer/Thr antibodies, followed by fluorescence-labeled second antibodies. Phosphorylation of MBP-30aa was observed on a protein chip, and this phosphorylation was inhibited by the PKC inhibitor (GF109203X). These results indicate the potential of PKCβ protein chip as a high-throughput screening tool in the screening of depigmenting agents.     

高通量蛋白质组学分析研究进展

基于质谱的蛋白质组学技术已经日趋成熟,可以对细胞和组织中的成千上万种蛋白质进行全面的定性和定量分析,逐步实现“深度覆盖”。随着生物医学日益增长的大队列蛋白质组学分析需求,如何在保持较为理想的覆盖深度下实现短时间、快速的“高通量”蛋白质组学分析已成为当前亟需解决的关键问题之一。常规的蛋白质组学分析流程通常包括样品前处理、色谱分离、质谱检测和数据分析。该文从以上4个方面展开介绍近10年以来高通量蛋白质组学分析技术取得的一系列研究进展,主要包括:(1)基于高通量、自动化移液工作站的蛋白质组样品前处理方法;(2)基于微升流速液相色谱与质谱联用的高通量蛋白质组检测方法;(3)利用灵敏度高、扫描速度快的质谱仪实现短色谱梯度分离下蛋白质组深度覆盖的分析方法;(4)基于人工智能、深度神经网络、机器学习等的蛋白质组学大数据分析方法。此外,对高通量蛋白质组学面临的挑战及其发展进行展望。总而言之,预期在不久的将来高通量蛋白质组学技术将会逐步“落地转化”,成为大队列蛋白质组学分析的利器。     

Functional proteomics: application of mass spectrometry to the study of enzymology in complex mixtures

This review covers recent developments in mass spectrometry-based applications dealing with functional proteomics with special emphasis on enzymology. The introduction of mass spectrometry into this research field has led to an enormous increase in knowledge in recent years. A major challenge is the identification of “biologically active substances” in complex mixtures. These biologically active substances are, on the one hand, potential regulators of enzymes. Elucidation of function and identity of those regulators may be accomplished by different strategies, which are discussed in this review. The most promising approach thereby seems to be the one-step procedure, because it enables identification of the functionality and identity of biologically active substances in parallel and thus avoids misinterpretation. On the other hand, besides the detection of regulators, the identification of endogenous substrates for known enzymes is an emerging research field, but in this case studies are quite rare. Moreover, the term biologically active substances may also encompass proteins with diverse biological functions. Elucidation of the functionality of those—so far unknown—proteins in complex mixtures is another branch of functional proteomics and those investigations will also be discussed in this review.