Markus F. Templin

Protein microarray technology.

This review summarizes the major activities in the field of protein microarray technology. A short summary of the theoretical concepts of miniaturized ligand binding assays explains why such microspot assays represent the most sensitive approaches for capture-target assays. The main focus of this review is centered on the applications using miniaturized and parallelized protein binding assays which rely on the product formation between immobilized capture molecules and their corresponding target molecules which are present in the sample. These types of ligand binding assays are useful tools for protein identification, quantification and protein affinity studies. Protein identification and quantification assays have a great potential in the field of diagnostics and proteomics where many different protein markers which are present in complex samples have to be analyzed in parallel. Protein affinity assays can be used to analyze interactions between proteins such as antibodies, receptors or enzymes with other proteins, peptides, low molecular weight compounds, oligosaccharides or DNA. Different applications of protein microarray-based assays and their huge potential for diagnostic and proteomic approaches will be discussed.

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

Protein microarrays for diagnostic assays

Protein microarray technology has enormous potential for in vitro diagnostics (IVD). Miniaturized parallelized immunoassays are perfectly suited to generating a maximum of diagnostically relevant information from minute amounts of sample whilst only requiring small amounts of reagent. Protein microarrays have become well-established research tools in basic and applied research and the first products are already on the market. This article reviews the current state of protein microarrays and discusses developments and future demands relating to protein arrays in their role as multiplexed immunoassays in the field of diagnostics.

Miniaturised multiplexed immunoassays.

Miniaturised immunoassays are of general interest for applications that require the simultaneous determination of different parameters from a minute sample of material. Apart from planar microarray-based systems, bead-based flow cytometric approaches are well suited for the multiplexed detection of target molecules, especially when the number of parameters that have to be determined in parallel is limited.

Protein microarrays: catching the proteome

After the completion of the human genome sequencing project, DNA microarrays and sophisticated bioinformatics platforms give scientists a global view of biological systems. In todays proteome era, efforts are undertaken to adapt microarray technology in order to analyse the expression of a large number of proteins simultaneously and screen entire genomes for proteins that interact with particular factors, catalyse particular reactions, act as substrates for protein-modifying enzymes and/or as targets of autoimmune responses. In this review, we will summarise the current stage of protein microarray technology. We will focus on the latest fields of application for the simultaneous determination of a variety of parameters from a minute amount of sample. Future challenges of this cutting-edge technology will be discussed.

Protein microarrays and multiplexed sandwich immunoassays: what beats the beads?

Protein microarray technology allows the simultaneous determination of a large variety of parameters from a minute amount of sample within a single experiment. Assay systems based on this technology are currently applied for the identification, quantitation and functional analysis of proteins. Protein microarray technology is of major interest for proteomic research in basic and applied biology as well as for diagnostic applications. Miniaturized and parallelized assay systems have reached adequate sensitivity and hence have the potential to replace singleplex analysis systems. However, robustness and automation needs to be demonstrated before this technology will finally prove suitable for high-throughput applications. Miniaturized and parallelized sandwich immunoassays are the most advanced assays formats among the different protein microarray applications. Multiplexed sandwich immunoassays can be used for the identification of biomarkers and the validation of potential target molecules. In this review an overview will be given on the current stage of protein microarray technology with a special focus on miniaturized multiplexed sandwich immunoassays.

Identification of Dlk1-Dio3 imprinted gene cluster noncoding RNAs as novel candidate biomarkers for liver tumor promotion.

The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, suggesting a role for β-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and β-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.

Proteome wide screening using peptide affinity capture

MS‐based strategies are key technologies for identifying proteins in proteomic research. Despite significant improvements in recent years efficient fractionation processes of target analytes remain major bottlenecks in MS‐based protein analysis. Immunoaffinity‐based sample fractionation strategies have shown their potential for the enrichment of analyte peptides of interest, but only small numbers of analytes can be quantified in one experiment. The lack of appropriate capture reagents limits the application of immunoaffinity‐based approaches and only biased biomarker discovery approaches are possible. This perspective discusses the current status of immunoaffinity MS‐based approaches and introduces a novel concept that uses group specific anti‐peptide antibodies – Triple X Proteomics Antibodies – for the enrichment of signature peptides. Classes of peptides with identical termini can be fractionated based on TXP immunoaffinity enrichment steps and can subsequently be identified using established tandem MS procedures. Based on bioinformatic algorithms minimal sets of TXP epitopes can be specified, that cover a wide range of given proteome landscapes of one or even several different species. This opens the possibility to use a minimal number of TXP antibodies as a universal toolbox for general immunoaffinity‐based approaches in proteome analysis.

Secretion of matrix metalloproteinase 3 by expanded articular chondrocytes as a predictor of ectopic cartilage formation capacity in vivo.

OBJECTIVE Monolayer expansion of human articular chondrocytes (HACs) is known to result in progressive dedifferentiation of the chondrocytes and loss of their stable cartilage formation capacity in vivo. For an optimal outcome of chondrocyte-based repair strategies, HACs capable of ectopic cartilage formation may be required. This study was undertaken to identify secreted candidate molecules, in supernatants of cultured HACs, that could serve as predictors of the ectopic cartilage formation capacity of cells. METHODS Standardized medium supernatants (n = 5 knee cartilage samples) of freshly isolated HACs (PD0) and of HACs expanded for 2 or 6 population doublings (PD2 and PD6, respectively) were screened by a multiplexed immunoassay for 15 distinct interleukins, 8 matrix metalloproteinases (MMPs), and 11 miscellaneous soluble factors. Cartilage differentiation markers such as cartilage oligomeric matrix protein and YKL-40 were determined by enzyme-linked immunosorbent assay. HACs from each culture were subcutaneously transplanted into SCID mice, and the capacity of the chondrocytes to form stable cartilage was examined histologically 4 weeks later. RESULTS Whereas freshly isolated (PD0) HACs generated stable ectopic cartilage that was positive for type II collagen, none of the cell transplants at PD6 formed cartilaginous matrix. Loss of the ectopic cartilage formation capacity between PD0 and PD6 correlated with a drop in the secretion of MMP-3 to <10% of initial levels, whereas changes in the other investigated molecules were not predictive. Chondrocytes with MMP-3 levels of >or=20% of initial levels synthesized cartilaginous matrix, whereas those with low MMP-3 levels (<10% of initial levels) at PD2 failed to regenerate ectopic cartilage. CONCLUSION Loss of the capacity for stable ectopic cartilage formation in the course of HAC dedifferentiation can be predicted by determining the relative levels of MMP-3, demonstrating that standardized culture supernatants can be used for quality control of chondrocytes dedicated for cell therapeutic approaches.

Microarray technology: an increasing variety of screening tools for proteomic research.

Protein microarray technology allows the simultaneous determination of a large variety of parameters from a minute amount of sample within a single experiment. Assay systems based on this technology are currently used for the identification, quantitation and functional analysis of proteins that are of interest for proteomic research in basic and applied biology and for diagnostic applications. Such novel assays are also of major interest for the pharmaceutical industry, focusing on the identification of biomarkers and the validation of potential target molecules. Sensitivity, reproducibility, robustness and automation have to be demonstrated before this technology will be suitable for high-throughput applications.