Analysis of protein expression in cell microarrays: a tool for antibody-based proteomics.

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome.     

Global Versus Local Centrality in Evolution of Yeast Protein Network

It is shown here that in the yeast protein interaction network the global centrality measure (betweenness) depends on the protein evolutionary age (i.e., on historic contingency) more weakly than the local centrality measure (degree). This phenomenon is not observed in mutational duplication-and-divergence models. The network domains responsible for this difference deal with DNA/RNA information processing, regulation, and cell cycle. A selection vector can operate in these domains, which integrates the network activity and thus compensates for the process of mutational divergence. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Differential binding studies applying functional protein microarrays and surface plasmon resonance

A variety of different in vivo and in vitro technologies provide comprehensive insights in protein-protein interaction networks. Here we demonstrate a novel approach to analyze, verify and quantify putative interactions between two members of the S100 protein family and 80 recombinant proteins derived from a proteome-wide protein expression library. Surface plasmon resonance (SPR) using Biacore technology and functional protein microarrays were used as two independent methods to study protein-protein interactions. With this combined approach we were able to detect nine calcium-dependent interactions between Arg-Gly-Ser-(RGS)-His6 tagged proteins derived from the library and GST-tagged S100B and S100A6, respectively. For the protein microarray affinity-purified proteins from the expression library were spotted onto modified glass slides and probed with the S100 proteins. SPR experiments were performed in the same setup and in a vice-versa approach reversing analytes and ligands to determine distinct association and dissociation patterns of each positive interaction. Besides already known interaction partners, several novel binders were found independently with both detection methods, albeit analogous immobilization strategies had to be applied in both assays.     

蛋白质芯片在蛋白质组学研究中的作用

蛋白质芯片是以高度并行性、高通量、微型化和自动化为特点的蛋白质组检测技术。本文综述了蛋白质芯片在蛋白质组学研究中的多种作用,包括普通蛋白质芯片在微量蛋白质分离、蛋白质与蛋白质之间以及蛋白质与其他小分子间相互作用和蛋白质定量检测方面的作用,普通蛋白质芯片通过与质谱技术、生物传感器技术的结合而拓展其应用范围,以及蛋白质组芯片、活性的蛋白质芯片在蛋白质组学研究中应用的进展。     

Printing of protein microarrays via a capillary-free fluid jetting mechanism

Current proteomics experiments rely upon printing techniques such as ink jet, pin, or quill arrayers that were developed for the creation of cDNA microarrays. These techniques often do not meet the requirements needed for successful spotting of proteins to perform high-throughput, array-based proteomic profiling. Biological laser printing (BioLP) is a spotting technology that does not rely on solid pins, quill pins, or capillary-based fluidics. The non-contact mechanism of BioLP utilizes a focused laser pulse to transfer protein solutions, thereby eliminating the potential for orifice clogging, air bubbles, and unnecessary volume loss potentially encountered in commercially available spotting technologies. The speed and spot-to-spot reproducibility of BioLP is comparable to other techniques, while the minimum spot diameter and volume per printed droplet is significantly less at 30 microm and approximately 500 fL, respectively. The transfer of fluid by BioLP occurs through a fluid jetting mechanism, as observed by high-speed images of the printing process. Arraying a solution of BSA with subsequent immunodetection demonstrates the reproducible spotting of protein in an array format with CVs of <3%. Printing of the enzyme alkaline phosphatase followed by a positive reaction with a colorimetric substrate demonstrates that functional protein can be spotted using this laser-based printer.     

酵母双杂交技术及其在蛋白质组研究中的应用

蛋白质组学是后基因组时代出现的一个新兴的研究领域,它的主要任务是识别鉴定细胞,组织或机体的全部蛋白质,并分析蛋白质的功能及其模式。因此,揭示蛋白质组中蛋白质间的相互作用关系也是蛋白质组学的重要内容之一。酵母双杂交技术是用来检测蛋白质间是否相互作用的一个非常有效的手段,该技术在酵母蛋白质组研究中的初步成功应用,表明它有望在人类蛋白质且研究中发挥重要作用。     

Interaction networks for systems biology

Cellular functions are almost always the result of the coordinated action of several proteins, interacting in protein complexes, pathways or networks. Progress made in devising suitable tools for analysis of protein-protein interactions, have recently made it possible to chart interaction networks on a large-scale. The aim of this review is to provide a short overview of the most promising contributions of interaction networks to human biology, structural biology and human genetics.     

Integrating mass spectrometry of intact protein complexes into structural proteomics

MS analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other MS approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative MS approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly advancing area.     

Protein microarrays and their applications

In recent years, the importance of proteomic works, such as protein expression, detection and identification, has grown in the fields of proteomic and diagnostic research. This is because complete genome sequences of humans, and other organisms, progress as cellular processing and controlling are performed by proteins as well as DNA or RNA. However, conventional protein analyses are time-consuming; therefore, high throughput protein analysis methods, which allow fast, direct and quantitative detection, are needed. These are so-called protein microarrays or protein chips, which have been developed to fulfill the need for high-throughput protein analyses. Although protein arrays are still in their infancy, technical development in immobilizing proteins in their native conformation on arrays, and the development of more sensitive detection methods, will facilitate the rapid deployment of protein arrays as high-throughput protein assay tools in proteomics and diagnostics. This review summarizes the basic technologies that are needed in the fabrication of protein arrays and their recent applications.     

Localized network centrality and essentiality in the yeast–protein interaction network

It has been suggested that a close relationship exists between gene essentiality and network centrality in protein–protein interaction networks. However, recent studies have reported somewhat conflicting results on this relationship. In this study, we investigated whether essential proteins could be inferred from network centrality alone. In addition, we determined which centrality measures describe the essentiality well. For this analysis, we devised new local centrality measures based on several well‐known centrality measures to more precisely describe the connection between network topology and essentiality. We examined two recent yeast protein–protein interaction networks using 40 different centrality measures. We discovered a close relationship between the path‐based localized information centrality and gene essentiality, which suggested underlying topological features that represent essentiality. We propose that two important features of the localized information centrality (proper representation of environmental complexity and the consideration of local sub‐networks) are the key factors that reveal essentiality. In addition, a random forest classifier showed reasonable performance at classifying essential proteins. Finally, the results of clustering analysis using centrality measures indicate that some network clusters are closely related with both particular biological processes and essentiality, suggesting that functionally related proteins tend to share similar network properties.     

Advances in the development of human protein microarrays

Introduction: High-content protein microarrays in principle enable the functional interrogation of the human proteome in a broad range of applications, including biomarker discovery, profiling of immune responses, identification of enzyme substrates, and quantifying protein-small molecule, protein-protein and protein-DNA/RNA interactions. As with other microarrays, the underlying proteomic platforms are under active technological development and a range of different protein microarrays are now commercially available. However, deciphering the differences between these platforms to identify the most suitable protein microarray for the specific research question is not always straightforward.

Areas covered: This review provides an overview of the technological basis, applications and limitations of some of the most commonly used full-length, recombinant protein and protein fragment microarray platforms, including ProtoArray Human Protein Microarrays, HuProt Human Proteome Microarrays, Human Protein Atlas Protein Fragment Arrays, Nucleic Acid Programmable Arrays and Immunome Protein Arrays.

Expert commentary: The choice of appropriate protein microarray platform depends on the specific biological application in hand, with both more focused, lower density and higher density arrays having distinct advantages. Full-length protein arrays offer advantages in biomarker discovery profiling applications, although care is required in ensuring that the protein production and array fabrication methodology is compatible with the required downstream functionality.     

Bioinformatics analysis of mass spectrometry-based proteomics data sets

Proteomics has made tremendous progress, attaining throughput and comprehensiveness so far only seen in genomics technologies. The consequent avalanche of proteome level data poses great analytical challenges for downstream interpretation. We review bioinformatic analysis of qualitative and quantitative proteomic data, focusing on current and emerging paradigms employed for functional analysis, data mining and knowledge discovery from high resolution quantitative mass spectrometric data. Many bioinformatics tools developed for microarrays can be reused in proteomics, however, the uniquely quantitative nature of proteomics data also offers entirely novel analysis possibilities, which directly suggest and illuminate biological mechanisms.     

Protein microarrays: Reduced autofluorescence and improved LOD

In protein microarray performance, the choice of an appropriate surface is a crucial factor. Three‐dimensional substrates like nitrocellulose are known to have higher binding capacities than planar surfaces. Furthermore, they can enable the immobilization of proteins in a functional manner. One disadvantage of today's nitrocellulose‐based microarrays is the high background fluorescence, which can interfere with the detection of low‐abundance proteins. We have developed an innovative black nitrocellulose membrane‐based protein microarray that exhibits low autofluorescence in combination with increased sensitivity and improved LOD (limit of detection). The applicability of the novel material was demonstrated with main focus on reversed‐phase microarray experiments. In comparison to various commercially available microarrays, a higher sensitivity in regard to the spotted protein was achieved. In contrast to other porous nitrocellulose‐based microarrays, the black nitrocellulose provides a significant lower autofluorescence and background intensity.     

Reading ADP-ribosylation signaling using chemical biology and interaction proteomics

1. Download : Download high-res image (282KB)
2. Download : Download full-size image

     

Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules

Affibody molecules, 58-amino acid three-helix bundle proteins directed to different targets by combinatorial engineering of staphylococcal protein A, were used as capture ligands on protein microarrays. An evaluation of slide types and immobilization strategies was performed to find suitable conditions for microarray production. Two affibody molecules, Z(Taq) and Z(IgA), binding Taq DNA polymerase and human IgA, respectively, were synthesized by solid phase peptide synthesis using an orthogonal protection scheme, allowing incorporation of selective immobilization handles. The resulting affibody variants were used for random surface immobilization (through amino groups) or oriented surface immobilization (through cysteine or biotin coupled to the side chain of Lys58). Evaluation of the immobilization techniques was carried out using both a real-time surface plasmon resonance biosensor system and a microarray system using fluorescent detection of Cy3-labeled target protein. The results from the biosensor analyses showed that directed immobilization strategies significantly improved the specific binding activity of affibody molecules. However, in the microarray system, random immobilization onto carboxymethyl dextran slides and oriented immobilization onto thiol dextran slides resulted in equally good signal intensities, whereas biotin-mediated immobilization onto streptavidin-coated slides produced slides with lower signal intensities and higher background staining. For the best slides, the limit of detection was 3 pM for IgA and 30 pM for Taq DNA polymerase.     

Characterization of the EGFR interactome reveals associated protein complex networks and intracellular receptor dynamics

Growth factor receptor mediated signaling is meanwhile recognized as a complex signaling network, which is initiated by recruiting specific patterns of adaptor proteins to the intracellular domain of epidermal growth factor receptor (EGFR). Approaches to globally identify EGFR‐binding proteins are required to elucidate this network. We affinity‐purified EGFR with its interacting proteins by coprecipitation from lysates of A431 cells. A total of 183 proteins were repeatedly detected in high‐resolution MS measurements. For 15 of these, direct interactions with EGFR were listed in the iRefIndex interaction database, including Grb2, shc‐1, SOS1 and 2, STAT 1 and 3, AP2, UBS3B, and ERRFI. The newly developed Cytoscape plugin ModuleGraph allowed retrieving and visualizing 93 well‐described protein complexes that contained at least one of the proteins found to interact with EGFR in our experiments. Abundances of 14 proteins were modulated more than twofold upon EGFR activation whereof clathrin‐associated adaptor complex AP‐2 showed 4.6‐fold enrichment. These proteins were further annotated with different cellular compartments. Finally, interactions of AP‐2 proteins and the newly discovered interaction of CIP2A could be verified. In conclusion, a powerful technique is presented that allowed identification and quantitative assessment of the EGFR interactome to provide further insight into EGFR signaling.     

High-throughput proteomics using antibody microarrays

Antibody-based microarrays are a novel technology that hold great promise in proteomics. Microarrays can be printed with thousands of recombinant antibodies carrying the desired specificities, the biologic sample (e.g., an entire proteome) and any specifically bound analytes detected. The microarray patterns that are generated can then be converted into proteomic maps, or molecular fingerprints, revealing the composition of the proteome. Using this tool, global proteome analysis and protein expression profiling will thus provide new opportunities for biomarker discovery, drug target identification and disease diagnostics, as well as providing insights into disease biology. Intense work is currently underway to develop this novel technology platform into the high-throughput proteomic tool required by the research community.     

Proteomic analysis of interactors for yeast protein arginine methyltransferase Hmt1 reveals novel substrate and insights into additional biological roles

Protein arginine methylation is a PTM catalyzed by an evolutionarily conserved family of enzymes called protein arginine methyltransferases (PRMTs), with PRMT1 being the most conserved member of this enzyme family. This modification has emerged to be an important regulator of protein functions. To better understand the role of PRMTs in cellular pathways and functions, we have carried out a proteomic profiling experiment to comprehensively identify the physical interactors of Hmt1, the budding yeast homolog for human PRMT1. Using a dual‐enzymatic digestion linear trap quadrupole/Orbitrap proteomic strategy, we identified a total of 108 proteins that specifically copurify with Hmt1 by tandem affinity purification. A reverse coimmunoprecipitation experiment was used to confirm Hmt1's physical association with Bre5, Mtr4, Snf2, Sum1, and Ssd1, five proteins that were identified as Hmt1‐specific interactors in multiple biological replicates. To determine whether the identified Hmt1‐interactors had the potential to act as an Hmt1 substrate, we used published bioinformatics algorithms that predict the presence and location of potential methylarginines for each identified interactor. One of the top hits from this analysis, Snf2, was experimentally confirmed as a robust substrate of Hmt1 in vitro. Overall, our data provide a feasible proteomic approach that aid in the better understanding of PRMT1's roles within a cell.     

Application of sector protein microarrays to clinical samples

Many protein functions are conferred by posttranslational modifications, which allow proteins to perform specific cellular tasks. Protein microarrays enable specific detection of posttranslational modifications not attainable by gene arrays. Reverse-phase protein microarrays have been widely adopted for use with clinical biopsy specimens because they have many advantages including highly reproducible printing of cellular lysates onto array surfaces, buit-in dilution curves, and direct detection using one antibody per analyte. This results in high-sensitivity, broad dynamic range, and favorable precision. Reverse-phase arrays have been restricted to a one slide/one antibody format. Although this is suitable for analyzing treatment effects over populations of samples, it is not well suited to individual patient assessments. One means of reaching this goal is the sector array format. Through the sector array, multiple antibody probes can be multiplexed on a single slide containing replicate immobilized aliquots from one patient. Thus, on one slide, a complete set of analytes can be characterized and used to support a therapy decision. This article describes a method for constructing sector arrays and demonstrates feasibility and adequate sensitivity applied to apoptosis related pathways.