The Affinomics programme aims to leverage existing efforts in Europe to generate large-scale resources of validated protein-binding molecules (binders) as affinity reagents for characterisation of the human proteome and to apply them in comprehensive structural and functional analyses of protein expression, interactions and complexes. The project was preceded by the ProteomeBinders and AffinityProteome consortia.
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Proteome targets will be focused on five categories of inter-related human proteins involved in signal transduction, cell regulation and cancer, namely protein kinases, SH2 domain-containing proteins, protein tyrosine phosphatases, proteins somatically mutated in cancers and candidate cancer biomarkers. Binders to about 1000 protein targets will be made over the course of the programme.
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A high throughput, coordinated production pipeline for antigens and binders will be established. Target antigens will be expressed in three forms, as folded full-length proteins or domains, as large peptide fragments (PrESTs) based on low homology to other human proteins and as small peptides, in some cases phosphorylated. Binder types to be generated include affinity-purified polyclonal antibodies, monoclonal antibodies, recombinant antibody fragments and non-immunoglobulin scaffolds.
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An important aspect will be the development of highly efficient next generation recombinant selection methods, based on phage, cell and ribosome display, capable of producing high quality binders at greater throughput and lower cost than hitherto. Systems and procedures for thorough binder validation and quality control will be established. The affinity reagents will be applied in advanced innovative and sensitive technologies for specific detection of target proteins and interacting protein complexes in cells, tissues and fluids, for improved understanding of protein function and new classes of diagnostic assays.
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For more detailed information, click on the map of the consortium.

Antibodies and other binder types are crucial for any proteome analysis. Although many tens of thousands of antibodies are available from commercial sources and academic institutions (see antibodypedia, for example), quality and reproducibility of analyses performed with these molecules vary substantially. Antibodies exhibit huge differences in specificity and affinity, including binders that target the same protein. Worse, even binders that are supposedly from the same source showed significant lot-to-lot variation.
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One reason for variation could be the actual assay; an antibody perfoming very well on Western blots may not meet the quality standards of pull-down experiments or microarray analyses, and vice versa. Another point, however, that is contributing significantly to low reproducibility is a lack of commonly accepted performance parameters and tests for their definition This is made worse by the fact that currently most binders are ill-described. Consequently, one cannot be sure, if a binder is exactly the molecule that was used in assays before.
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Initiated by Andrew Bradbury, 101 scientists of the Affinomics consortium and beyond have had some thoughts about the matter. A simple solution to the problem of how to make sure that everybody is really using the very same binder molecules in their experiments would be a sequence verification of each binder. While relatively easily achievable for recombinant binders and monoclonal antibodies, it is more difficult to establish for polyclonal binders, for example. A more important obstacle for an implementation of such a scheme, however, could be the reluctance of antibody producers to share the sequences of their molecules, since this would make available their good binders to the entire scientific (and commercial) community for free.

Protein profiling was performed on gastric cancer tissue samples. Sixteen pairs of postoperative gastric adenocarcinomas and adjacent non-cancerous control tissues were analysed on microarrays that contain 813 antibodies targeting 724 proteins. Only 17 proteins were found to be differentially regulated, much fewer molecules than the number identified when comparing tumour to healthy control tissues. Insulin-like growth factor-binding protein 7 (IGFBP7), S100 calcium binding protein A9 (S100A9), interleukin-10 (IL-10) and mucin 6 (MUC6) exhibited the most profound variations. For an evaluation of the proteins’ capacity for discriminating gastric cancer, a Receiver Operating Characteristic curve analysis was performed. For confirmation, immunohistological analyses were done on samples prepared from another cohort of patients with gastric cancer.

Figure legend: Typical results of immunohistochemical analyses. Identified marker molecules were validated by immunohistochemistry on an independent set of tumour tissues and stomach samples from donors who had no cancer. Dark brown colour is indicative of the presence of the respective proteins.

FINISHED PROJECT:
Definition of a serum marker panel for glioblastoma discrimination and identification of interleukin 1β in the microglial secretome as a novel mediator of endothelial cell survival induced by C-reactive protein

Glioblastoma (GBM) is the most common malignant adult primary brain tumour. We profiled 724 cancer-associated proteins in sera of healthy individuals (n = 27) and GBM (n = 28) using an antibody microarray. While 69 proteins exhibited differential abundance in GBM sera, a three-marker panel (LYAM1, BHE40 and CRP) could discriminate GBM sera from that of healthy donors with an accuracy of 89.7% and p = 0.0001.

The high abundance of C-reactive protein (CRP) in GBM sera was confirmed in 264 independent samples. High levels of CRP protein were seen in GBM but without a change in transcript levels suggesting a non-tumoral origin. Glioma-secreted Interleukin 6 (IL6) was found to induce hepatocytes to secrete CRP, involving the JAK-STAT pathway. The culture supernatant from CRP-treated microglial cells induced endothelial cell survival under nutrient-deprivation condition involving the CRP-FcγRIII signalling cascade. Transcript profiling of CRP-treated microglial cells identified Interleukin 1β (IL1β) present in the microglial secretome as the key mediator of CRP-induced endothelial cell survival. IL1β neutralization by antibody-binding or siRNA-mediated silencing in microglial cells reduced the ability of the supernatant from CRP-treated microglial cells to induce endothelial cell survival. Our study identifies a serum based three-marker panel for GBM diagnosis and provides leads for developing targeted therapies.
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About 70% of newly diagnosed cases of bladder cancer are low-stage, low-grade, non muscle-invasive. Standard treatment is transurethral resection. About 60% of the tumours will recur, however, and in part progress to become invasive. Therefore, surveillance cystoscopy is performed after resection. In the USA and Europe alone, about 54,000 new patients per year undergo repeated cystoscopies over several years, who do not experience recurrence. Analysing in a pilot study resected tumours from patients with and without local recurrence after a period of five years, we identified 255 proteins with significantly differential abundance. Most are involved in the regulation and execution of apoptosis and cell proliferation. A multivariate classifier was constructed based on 20 proteins. It facilitates the prediction of recurrence with a sensitivity of 80% and a specificity of 100%. As a measure of overall accuracy, the area under the curve (AUC) value was found to be 91%. After validation, such a signature could help to adjust the treatment scheme and the rigidity of surveillance. This could dramatically reduce surveillance cost and simultaneously improve the patients’ outcome substantially.

Figure legend: Strong repression of the TGF-beta signalling pathway in recurrent cancer. Affected proteins are labelled in green or red if their expression was lower or higher, respectively, in recurrent rather than non-recurrent cancer.

Purpose: Plasma samples from a multi-centric case control study on lymphoma were analysed for the identification of proteins useful for diagnosis.
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Experimental design: The protein content in the plasma of 100 patients suffering from the three most common B-cell lymphomas and 100 control samples was studied with antibody microarrays composed of 810 antibodies that target cancer-associated proteins. Sample pools were screened for an identification of marker proteins. Then, the samples were analysed individually to validate the usability of these markers.
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Results: More than 200 proteins with disease-associated abundance changes were found. The evaluation on individual patients confirmed some molecules as robust informative markers while others were inadequate for this purpose. In addition, the analysis revealed distinct subgroups for each of the three investigated B-cell lymphoma subtypes. With this information, we delineated a classifier that discriminates the different lymphoma entities.
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Conclusions and clinical relevance: Variations in plasma protein abundance permit discrimination between different patient groups, defining a diagnostic as well as differential-diagnostic potential. Beside this, methodological aspects were critically evaluated such as the value of sample pooling for the identification of biomarkers that are useful for a diagnosis on individual patients.

Definition of disease-specific protein profiles with sample pools. Correspondence analysis resulted in a biplot of both differentially abundant proteins and the samples; the two axes represent the first and second principal component, respectively. Sample pools are depicted as squares that are coloured according to disease status; black spots stand for differentially expressed proteins.

The goal of the project was the development of a real-time fluorescence detection system of protein-ligand interactions. The systems allows the effective combination of different molecular biology procedures with ultra-sensitive, multiplexed detection, the final objective being a real-time analysis of biomolecular interactions for virtually any kind of biomolecules under various conditions.
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In recent years, the detailed study of biomolecular interactions has become a very important and growing element of biomedical research. This increased interest has been driven by the appreciation that a deep understanding of protein interactions is fundamental to both the study of disease and the elucidation of the action of small molecule drugs or biopharmaceuticals. Protein-based pharmaceuticals are currently enjoying great commercial success and are accounting for a growing proportion of the pharmaceutical industry’s development portfolio.
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Although some detection schemes and relevant equipment have been developed for the measurement of biomolecular interactions, a lot of progress remains to be done in terms of sensitivity, multiplexing, time-scale and particularly quantification, especially for low abundance molecules.
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The capabilities of the fluorescence-based detection was used mainly for the quantitative measurement of protein expression in patient material by means of antibody microarrays as well as quantitative analysis of protein-protein and protein-drug interactions in real-time.

FINISHED PROJECT:
Immunoassay-based proteome profiling of 24 pancreatic cancer cell lines

Pancreatic ductal adenocarcinoma is one of the most deadly forms of cancers, with a mortality that is almost identical to incidence. The inability to predict, detect or diagnose the disease early and its resistance to all current treatment modalities but surgery are the prime challenges to changing the devastating prognosis. Also, relatively little is known about pancreatic carcinogenesis.
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In order better to understand relevant aspects of pathophysiology, differentiation, and transformation, we analysed the cellular proteomes of 24 pancreatic cancer cell lines and two controls using an antibody microarray that targets 741 cancer-related proteins. In this analysis, 72 distinct disease marker proteins were identified that had not been described before. Additionally, categorizing cancer cells in accordance to their original location (primary tumour, liver metastases, or ascites) was made possible.
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A comparison of the cells’ degree of differentiation (well, moderately, or poorly differentiated) resulted in unique marker sets of high relevance. Last, 187 proteins were differentially expressed in primary versus metastatic cancer cells, of which the majority is functionally related to cellular movement.
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Figure legend: Immunohistochemical analysis of five cell lines. Using fluorescently stained antibodies against collagen-XV-α1 and tetraspanin-6, only cell line MDA-Panc-28 exhibited strong signal intensities, which is consistent with the microarray data and indicates the specific expression of the two proteins in cells of acinar origin. The other four cells are of ductal lineage.

The process of extracting comprehensive proteome representations is a crucial step for many proteomic studies. We developed two single-step extraction buffers - Mix 1 and Mix 2 - for the isolation of proteins from mammalian tissues under native conditions in an effective and reproducible manner.
Protein extractions were performed from cell lines BxPC-3 and SU.86.86, rat organs (pancreas, liver, heart and lung) and human pancreatic cancer tissues. In comparison to several buffer systems that contained individual non-ionic or zwitterionic detergents as well as to commercial extraction buffers, the two buffer systems were used. Each contains a detergent cocktail that includes at least one polymeric phenylethylene glycol, a long-chain amidosulfobetaine, cholate and a zwitterionic detergent. Extracts obtained with the various buffer systens were analysed for protein quantity and quality. The two detergent cocktails exhibited superior extraction capacity. Also, they demonstrated a substantially higher recovery of membrane and compartmental proteins as well as much better preservation of protein functionality. In addition, they did not interfere with subsequent analysis steps such as labelling, a problem that was observed with other buffers. In Western blot and antibody microarray assays, they out-performed the other buffer systems, demonstrating their usefulness for various types of proteomic studies.

We are developing systems for the analysis of complex protein extracts on antibody-microarrays. Currently, the availability of specific and highly affine antibodies is a limiting factor in such studies. In collaboration with the Department of General Surgery at the University of Heidelberg and Jörg Hagen of the Proteomics Unit at Merck in Darmstadt, we worked at the establishment of techniques for selecting highly specific antibodies.
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Central issue to the project was the isolation of highly specific antibodies from phage display libraries. Antibodies isolated from phage display libraries are typically selected using purified antigens immobilised on plastic surfaces. In general, however, selection requires laborious subtraction protocols to avoid the selection of irrelevant antibodies. In spite of such elaborate pre-incubations, selection of antibodies by cell panning is limited by background binding of non-specific phage and relatively low binding of specific phage. Also, good binders frequently get lost during the process. Because of the growing need for antibodies to be used in early, preventive cancer diagnostics and tumour specific therapeutics, we established a process that circumvents the above mentioned problems and offers an opportunity selectively to identify highly specific and affine antibodies. In addition, since all steps occur in vitro, the technology allows automatisation of the entire process, which is critical for large-scale applications.

High-specificity affinity reagents (‘binders’) are essential probes for proteome research, enabling the detection and localisation of multiple proteins in tissues and fluids in health and disease through the application of binder-based technologies (affinity proteomics). This project linked the high-throughput production of quality-controlled, recombinant binding molecules of different types (antibody fragments, engineered scaffolds, aptamers) with advanced applications (capture microarrays, multidimensional fluorescence imaging, single-molecule detection, intracellular knockdown) in the analysis of human proteome targets. 
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The partners were five established SMEs and five academic groups engaged in binder production and characterisation or protein detection technologies. The project aimed at resolving bottlenecks in high-throughput binder production and application technologies, including cost, throughput, automation and quality. The linkage of production, quality control and applications was exemplified by targeting proteins involved in signal transduction pathways. These pathways are implicated in many diseases. Also, drug development is actively evolving in this area. Therefore, access to high-quality binding reagents is particularly needed.
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The project benefited from close interaction with the FP6 Coordination Action ProteomeBinders, which commenced earlier and provided also a framework for activities such as the ongoing Affinomics project.

On the basis of transcriptional profiling experiments on DNA-microarrays and other results, some 900 genes of interest were selected. Antibodies for the respective proteins were generated in a collaboration with the company Eurogentec by selecting and synthesising appropriate peptide sequences. The peptides were used for the immunisation of rabbits, from which affinity-purified antibodies were obtained.
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Further characterisations were performed to define specificity and affinity of the molecules.This process provided us with an initial set of some 650 antibodies that have been used in a large number of experiments, validating their value and performance. We continue using them as probes on microarrays and beyond for analyses of protein expression variations. Further antibodies with particular characteristics are being produced and checked for their suitability for cancer diagnosis.
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With the existing microarray, we pursue biomedical studies toward an early and accurate diagnosis on tissues, cells and various forms of body fluids. In addition, we are aiming at understanding protein-based communication between the cells of the tumour microenvironment. Analyses are under way comparing transcriptional changes and the actual variations at the protein level. Also other applications are being worked at.
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Apart from the production of antibodies by classical means, other procedures for the isolation of antibodies are being pursued. Activities in the group are mainly based on display libraries. In addition, we collaborate with partners within the Affinomics consortium, who actively produce binders of different formats.

Schröder et al. (2010) Mol. Cell. Prot. 9, 1271-1270.

                

Proteins are the major class of effector molecules in cellular systems. For the identification of functional differences between normal and diseased tissues, a reliable analysis of their protein content is essential. Reproducible isolation and fractionation of intact proteins are important in this respect. The complexity of proteins in structure and concentration, their close interaction as well as their instability represent major challenges. For isolation from tissues, also the destruction of the cell-cell and cell-matrix connections within a tissue without affecting protein quality is a critical factor. We compared different processes for a compartmental protein preparation from pancreatic tissues, using matching normal and tumour tissue samples from the same patients. Pancreas is one of the most challenging tissues because of its high content of proteases.
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Success of the different procedures varied strongly. Based on a scheme of slicing the tissues and a subsequent isolation of the cells, we established a workflow for the extraction in a reproducible manner of fractions of cytosolic proteins, membrane and organelle proteins, nuclear proteins and cytoskelatal filaments. The tissue slices also allow for a representative confirmation of the individual samples’ cellular status by histochemical processes and a proper separation or mixing of cellular material from across a tumour if required.

The suitability of simple and inexpensive detection approaches for highly sensitive antibody microarray analysis was examined. N-hydroxysuccinimidyl ester (NHS) and Universal Linkage System (ULS) based fluorescein and biotin labels used as tags for subsequent detection with anti-fluorescein and extravidin, respectively, as well as fluorescent dyes were applied for analysis of blood plasma. Parameters modifying strongly the performance of microarray detection such as labelling conditions, incubation time, concentrations of anti-fluorescein and extravidin and extent of protein labelling were analysed and optimised. Indirect detection strategies whether based on NHS- or ULS-chemistries strongly outperformed direct fluorescent labelling and enabled detection of low abundant cytokines with many dozen-fold signal-to-noise ratios.

We examined the limitations of existing microarray immunoassays and investigated how best to optimise them using theoretical and experimental approaches. A key physicochemical limitation of microarray immunoassays is a strong dependence of antibody microspot kinetics on the mass flux to the spots. We analysed theoretically and experimentally the effects of microarray design parameters (incubation vessel geometry, incubation time, stirring, spot size, antibody-binding site density, etc.) on microspot reaction kinetics and sensitivity.
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Using a two-compartment model, the quantitative descriptors of the microspot reaction were determined for different incubation and microarray design conditions. This analysis revealed profound mass transport limitations in the observed kinetics, which may be slowed down as much as hundreds of times compared with solution kinetics.
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The data obtained were considered with relevance to microspot assay diffusional and adsorptive processes, enabling us to validate some of the underlying principles of the antibody microspot reaction mechanism and provide guidelines for optimal microspot immunoassay design. For assays optimised to maximize the reaction velocity, we could demonstrate sensitivities in the attomolar and low femtomolar ranges.

Antibody microarrays will have an enormous impact on the functional analysis of cellular activity and regulation, especially at the level of protein expression and protein-protein interaction. The array surface is bound to have a tremendous influence on the findings from such studies.
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Apart from the basic issue of how to attach antibodies optimally without affecting their function, it is also important that the cognate antigens, applied within a complex protein mixture, all bind to the arrayed antibodies irrespective of their enormous variety in structure.
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We analysed various factors in the production of antibody microarrays on glass support – the modification of the glass surface, kind and length of crosslinkers, composition and pH of the spotting buffer, blocking reagents, antibody concentration, storage procedures, etc. – in order to evaluate their effect on array performance. Altogether, data from more than 1200 individual array experiments was taken into account. In addition to home-made slides, also commercially available systems were included in the analysis. The results of this work can be found in the manuscripts listed below.
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Further improvements were made more recently and are documented on the current webpage.