Genome Analysis (B070)
Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580
D-69120 Heidelberg, Germany.
Tumour Analysis / Proteomics
Technologies / Epigenetics
|Antibody microarrays||Antibody selection||Expression profiling||Protein microarrays||Peptide synthesis|
| Detailed protocols
||Pancreatic cancer||In situ expression||Automated purification|
||Peptide-based selection||Affinomics||Bladder cancer|| ARTAMIS
||Panning selection||Lymphoma||Single molecule detection||Archive|
As an immediate consequence of the large-scale genomic sequencing efforts, a strong interest has emerged in analysing the function of the DNA-encoded information on a similarly global scale. However, many aspects of modulation and regulation of cellular activity cannot be investigated on the level of nucleic acids but require an analysis of the proteome. Several studies demonstrated poor correlation of mRNA and protein levels. This is due to post-transcriptional control of protein translation, a number of post-translational modifications of protein as well as protein degradation by proteolysis. Recent estimations suggest that there are more than 200 types of protein modification. The proportion and importance of protein modification is reflected by the fact that 5% to 10% of mammalian genes encode for proteins that modify other proteins.
Consequently, the complexity in the human proteome is expected to range from a hundred thousand to several million different protein molecules. With respect to data interpretation, the situation is additionally complicated by the facts that no function is known for very many of the predicted proteins of multicellular organisms and that the dynamic range of protein expression can be very large indeed.
With two-dimensional gel-electrophoresis and various processes based on mass spectrometry, powerful techniques exist for the analysis of proteins of an organism or tissue. Yeast-two-hybrid analyses also permit global studies for the identification of interaction partners. Nevertheless, many other, possibly even more powerful methods are prerequisite to approaching the world of protein analysis in a manner similar to what is already possible for studies at the level of nucleic acids, and beyond.
Protein microarrays have an enormous potential of developing into a tool that will allow at the protein level the type of global characterisation of molecule mixtures. Knowledge of genomic sequences and transcriptional profiles do not allow a reliable description of actual protein expression, let alone an analysis of the proteins' structure and biochemical activities, as well as variations thereof, or a quantitative examination of protein-protein interactions. This kind of information, however, is important for a molecular characterisation of physiological or developmental cellular stages and has a broad biotechnical and medical potential. Utilising recently developed processes, we are able to perfom such analyses even on a large-scale with nevertheless high reproducibility, a near-single-molecule sensitivity and an accuracy that is as good as ELISA-based assays or DNA-microarray analyses.
Utilising antibody microarrays, we pursue the analysis of studying variations in actual protein expression, isoform occurrence and other structural variations such as modifications in tissues and body liquids. Initially, basic technical processes were studied in detail such as appropriate surfaces, the effect of kinetics and mass transport and well as labelling procedures, for example, in order to establish a working system. Detailed protocols are available that allow reproducible and reliable analysis of expression variations on complex protein extracts down to attomolar concentrations. Antibodies generation and selection was and is performed in collaborations with company partners as well as EU-funded initiatives that aim at the creation of well-characterised and specific antibodies or other binders (ProteomeBinders; AffinityProteome; Affinomics). In addition, improvements of the preparation of protein extracts proved crucial for success, in particularly for analyses of samples from tissue and cell culture. The current set-up was and is used in various projects (e.g., MolDiagPaca; Drop-Top; PaCaNet; ARTAMIS) for actual measurements, frequently combining the information on protein levels with other data. Also, quantification of the results is performed, either by actual counting of individual molecules or by an analysis of dissociation parameters.
We utilise a process of protein microarrays containing (in part full-length) molecules for the investigation of protein-protein interactions in a quantitative manner. Microarray production is done by in situ synthesis by an transcription/translation process on the microarrays. Protein interaction of all kinds as well as the influence of co-factors such as small molecules can be studied this way by detecting the signal intensities obtained upon incubation with a protein sample. Again, the usefulness of the data can be improved by obtaining information on the dissociation behaviour of interacting molecules or their actual number. This was pursued in the ARTAMIS project.
The Affinomics programme aims to leverage existing efforts in
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.
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.
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.
For more detailed information, click on the map of the consortium.
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 links 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.
The partners are five established SMEs and five academic groups engaged in binder production and characterisation or protein detection technologies. The project aims 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 will be exemplified by targeting proteins involved in signal transduction pathways, which are implicated in many diseases and where the availability of high-quality binding reagents is particularly needed and drug development is actively evolving.
The project benefits from close interaction with the FP6 Coordination Action ProteomeBinders, which commenced earlier and provided a framework for activities such as AffinityProteome.
Alhamdani et al. (2009) Genome Med. 1, 68.
Schröder et al. (2010) Mol. Cell. Prot. 9, 1271-1270.
|RECENTLY FINISHED PROJECT:
A European infrastructure of ligand binding molecules against the human proteome
|Creation of an antibody microarrays for the analysis of the expression of cancer associated proteins|
basis of transcriptional
profiling experiments on DNA-microarrays and other results, some 900
genes of interest were selected. Antibodies for
proteins were generated in a collaboration with the company Eurogentec by
synthesising appropriate peptide sequences.
The peptides were used for the immunisation of rabbits, from which
affinity-purified antibodies were obtained. Further characterisations
were performed to define specificity and affinity of the
molecules.This process provided us
with an initial set
some 800 antibodies
that are being used as probes on microarrays for analyses of protein
expression variations. Currently, further
antibodies with particular
characteristics are being produced and checked for their suitability
With the existing microarray, we pursue biomedical studies toward an early and accurate diagnosison tissues and body fluids. In addition, analyses are under way comparing transcriptional changes and the actual variations at the protein level. Also other applications are being worked at.
Apart from the production of antibodies by classical means, other procedures for the isolation of antibodies are worked at. Activities in the group are mainly based on display libraries, such as described below. In addition, we collaborate with partners within the Affinomics, AffinityProteome and ProteomeBinders consortia, who actively produce binders of different formats.
Schröder et al. (2010) Mol. Cell. Prot. 9, 1271-1270.
|Selection of highly specific antibodies for the identification of molecular differences in the proteome of normal and tumour cells|
developing systems for
the analysis of complex protein extracts on antibody-microarrays.
availability of specific and highly affine antibodies is a limiting
such studies. In
with the Department
of General Surgery at the
Central issue to the project is the isolation of highly specific antibodies from phage display libraries. Antibodies isolated from phage display libraries have typically been 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 high 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.
Hoheisel (2005) PCT / EP2005 / 008431.
procedure for the isolation of proteins at near-native conditions from
mammalian tissue for proteomic analysis
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.
Alhamdani et al. (2010) J. Prot. Res. 9, 963-971.
Comparison of protein extraction efficacy using the two newly established buffer systems and the best performing commercial procedure in the analysis. Typical results with proteins from different cell compartments are shown.
protocols for expression profiling by antibody microarrays
As a multiplexing technique, antibody microarrays facilitate the highly parallel detection of hundreds of different analytes from very small sample volumes of only few microliters. This is combined with a high sensitivity in the picomolar to femtomolar range, which is similar to the sensitivity of ELISA, the gold standard for protein quantification. In order to obtain such sensitivities in a robust and reproducible manner for sets of several hundreds of analytes, it is essential to optimise the experimental layout, sample handling, labelling and incubation as well as data processing steps.
Based on earlier work, we continuously developed the processing of microarrays and protein samples. In the publications listed below, we present in detail our antibody microarray protocols for multiplexed expression profiling studies, which permit the analysis of the abundance of more than 800 proteins in plasma, urine and tissue samples.
Due to the success of DNA-microarrays and the growing numbers of available protein expression clones, protein microarrays become more and more popular for the high-throughput screening of protein interactions. However, the widespread applicability of protein microarrays for this and other applications is currently still hampered by the large effort associated with their production. Beside the requirement for a protein expression library, the expression and purification of the proteins themselves and the lacking stability of many proteins remains the bottleneck.
As part of the MolTools project, we established a process that allows the generation of protein microarrays from unbound DNA template molecules on the chip. It comprises the spotting of a DNA template in a first spotting and the transfer of a cell-free transcription and translation mix on top of the same spot in a second spotting run. Using wildtype GFP as a model protein, we demonstrated the time and template dependence of this coupled transcription and translation and showed that enough protein is produced to yield signals that are comparable to 300 µg/ml of spotted protein. Plasmids as well as unpurified PCR-products can be used as templates and as little as 35 fg of PCR-product (~22,500 molecules) are sufficient for the expression of full-length wildtype GFP. Both aminopropyltriethoxysilane and Ni-chelate surfaces can be used for capture of the newly synthesized proteins. As a surprise, we observed that also Ni-chelate coated slides are binding the his-tagged proteins in an unspecific manner.
We adapted the system to the high-throughput expression of libraries by designing a single primer pair harbouring promoter, ribosomal binding site and terminator sequences for an on the chip expression of a multitude of such PCR-products. Utilising full-length cDNA libraries of overall 16,000 clones, we are aiming at the production of a comprehensive microarrays of all proteins of the human genome (assuming one gene coding for one protein). Based on such arrays, quantitative measurements of protein-protein interactions and the influence of various compounds are performed.
Utilising the capability of detecting the interaction of individual molecules and therefore being able to count the actual number of interacting molecules, we can perform interaction studies in a really quantitative manner. Complementarily, we have acquired hardware as part of the ARTAMIS project that permits the detection of the dissociation behaviour of many interaction partners simultaneously.
real-time and multiplex analysis of biomolecular interactions (ARTAMIS)
The goal of the project is the development of a real-time fluorescence detection system of protein-ligand interactions. The systems will allow 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.
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.
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.
The outstanding capabilities of the fluorescence-based detection is 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.
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