5th ESF Workshop on Affinity Proteomics
14-16 March 2011
Alpbach, Austria

Organisers:

Draft Report

Summary

The ESF/AFFINOMICS Affinity Proteomics workshop, held from March 14-16th 2011 in Alpbach (Austria), was attended by 103 academic and industrial participants from Europe and the US. The focus was on the current state of affinity methods for proteome analysis. It covered a wide area, including the human atlas and proteome projects, the application of binders in cell signalling research, and complementarity to more conventional mass spectrometry proteomics. There was a focus on new technologies, from recombinant binder production methods to binder applications in protein imaging on the micro and macro scales, array-based analyses and microfluidics systems. In addition to 17 principal invited speakers, there were 6 short talks selected from submitted abstracts and 25 poster presentations. The programme consisted of two and a half days of talks, with time set aside for outdoor activities and continuing discussion during the early afternoon, a layout which proved very popular with all participants. The meeting was co-funded by the ESF Programme Frontiers of Functional Genomics and the EU Framework 7 collaborative project AFFINOMICS.

Scientific Content

The design of the meeting programme was to address recent developments in affinity proteomics, the characterisation of proteins on a genome-wide scale using specific binding reagents, such as antibodies, protein scaffolds, aptamers and peptides. In order to achieve this, there is an acknowledged need to establish comprehensive resources of well- characterised affinity binding reagents (a.k.a. binders) together with technologies for their application. The production of such resources also has important potential clinical applications in diagnostics and therapeutics, as well as providing a major post-genome challenge with significance for both basic and medical research. A central aspect is the systematic generation of binders by modern high throughput technologies, particularly recombinant methods. However, the technology of binder production is only one aspect; such projects also need to begin with a clear definition and prioritisation of target molecules. In the case of the EU projects linked to the workshop, AffinityProteome and AFFINOMICS, the focus is on cell signal transduction molecules, both for their intrinsic importance in all cellular activity and response, but also because of their frequent involvement in cancer as a result of mutation. A related area of medical importance where binders will be actively employed is in the definition of disease-specific biomarkers, particularly at the plasma level; it is likely that combinations of altered concentrations of several proteins will provide diagnostic ‘signatures’ which can have predictive value. Such developments and applications of binders will directly relate to personalised medicine and the individualisation of patient treatment, the new paradigm of healthcare. These topics were covered in different presentations through the meeting. In the opening session (Monday 14th March) Mathias Uhlén (Stockholm) described progress in the human atlas project, perhaps the most complete example to date of an affinity approach to defining the human proteome. Essentially this project will map the location of each encoded gene product, and has recently passed the halfway point of about 10,000 genes. There is still much to be done, and particularly where proteins are modified for activity or exist in multiple splice variants and isoforms. Nevertheless, important lessons are being learned about protein expression in different tissues and cell types, and indications of potentially useful biomarkers are emerging. This was followed by an account of the Human Proteome Project (HPP) being established by HUPO. The coordinator of the HPP, Pierre Legrain (Paris) described its gene-centric approach, similar to that of the protein atlas, and organisation around individual chromosomes, for which the proteomics will be tackled by groups in different countries. There was a lively discussion about this aspect of the project and the integration of different centres, which will be highly challenging.

In the following session (Tuesday 15th March), aspects of cell signalling proteomics were described by Gianni Cesareni (Rome), focusing on protein phosphatase networks, and by Lukas Huber (Innsbruck) who described use of tandem affinity purification and mass spectrometry to understand the scaffold signalling complexes of the MAPK pathway. These talks exemplified the use of complementary technologies, including chips, siRNA, and fluorescence microscopy, to define signalling networks and their regulation. A further contribution to the signal transduction analysis using binders was provided by Anke Prinz (Kassel) who gave a short talk demonstrating the intracellular effects of Designed Ankyrin Repeat Proteins (DARPins) on the MAPK pathway using the technique of Bioluminescence Resonance Energy Transfer (BRET), which is less well known than the FRET counterpart for fluorescence but provides similar dynamic information on protein-protein interactions. The intracellular action of binders (intrabodies) is likely to be a key area of future application.

The aspect of personalised medicine was then introduced by Klaus Lindpaintner (Newark DE), formerly of Roche and now director of antibody production at Strategic Diagnostics. He focused on the importance of protein biomarker discovery and the requirements of binders for the application to predictive healthcare, with insights into the role of industry. The personalised medicine theme was further developed in relation to cancer by Olli Kallioniemi (Helsinki), who described in detail a series of studies on a rare bone tumour, graphically illustrating the importance of treating the individual cancer by all available means, including experimental methods.

In an approach which caused considerable fascination, Mariliza Derveni (Cranfield, UK) proposed the use of specific binders in space exploration, particularly as a means to detect signs of life on other planets in our solar system, notably Mars. The concept is that antibodies to organic molecules associated with life on Earth will be used to construct an immunoassay device (Life Marker Chip) to be incorporated into future space probes. The problem will be partly to predict the types of molecules most likely to be present and the related one of which affinity reagents are best suited for such a purpose. This project will be watched with interest!

In a session which brought the meeting up to date on alternatives to conventional antibodies, Andreas Plückthun (Zurich) and Larry Gold (Boulder) described the DARPins and DNA aptamers respectively. The latter have been refined as SOMAmers, aptamers with high affinity due to slow off rates, of which over 1000 have now been prepared against the same number of human proteins for biomarker discovery, with particular progress in lung cancer among others. Finally different recombinant selection systems based on yeast and phage display were described by Andrew Bradbury (Los Alamos).

On day 3 (Wednesday 16th March), the theme of cell signalling and cancer was continued from the perspective of mass spectrometry by Bernhard Küster (Munich), using kinobeads to trap kinases and investigate the associated protein complexes. This represents another form of protein affinity purification using ligand rather than antibody. This approach has been used by his group to identify new kinase targets for existing inhibitors, such as the leukaemia drug Imatinib.

Ulf Landegren (Uppsala) took binder technology in the direction of protein-protein interaction with the proximity ligation assay, in which binders are complexed to DNA strands which can be ligated only when the binders are close together, and then amplified for sensitive visualised detection. The assay, which has been successfully commercialised by OLINK in Uppsala, has advanced to the stage of determination and digital recording of protein complexes of many types in plasma and in situ in tissue sections. It has been shown to be very useful for detection of changes during cell signalling, such as phosphorylation. A sensitive assay for prostate cancer using 5 binders to target complexes called prostasomes in patient plasma is under development. Imaging of protein distribution in networks was taken up by Walter Schubert (Magdeburg) who described the toponome methodology or Multi-Epitope Ligand Cartography, in which antibodies are used to demonstrate protein clusters also through detection of co-localisation on repeated cycles of fluorescent staining, imaging and bleaching. This has led to target discovery in a number of diseases, including muscle disorders, tumours and neuropathic pain.

Several short talks then dealt with array type assay systems, including antibodies immobilised on bead suspension arrays (Fridtjof Lund-Johansen, Oslo; Jochen Schwenk, Stockholm), and on glass slides as complex antibody microarrays (Christoph Schröder, Heidelberg). These assays now allow thousands of protein estimations to be carried out in parallel and can be used for screening in a number of clinical settings. With their huge sample throughput potential, and the availability of relevant numbers of binders, these formats will be powerful biomarker discovery tools for the future.

The possibility of ‘individualising’ proteomics was described by Mike Taussig (Cambridge) in the context of protein microarrays. By using cell free protein production from DNA, it is possible to express proteins directly onto the surface of arrays using the techniques of PISA (Protein in situ Array) and DAPA (DNA to Protein Array). Knowledge of individual variation at the DNA level from next generation sequencing can thereby be converted directly into expressed proteomes for investigations of altered functions such as network interactions. This was also demonstrated using sophisticated microfluidic technology by Doron Gerber (Ramat Gan), who has designed his system called PING, in which proteins synthesised in situ from DNA by cell free transcription and translation were used to screen for virus-host protein interactions. This can be scaled potentially to whole proteome level screens using the microfluidic setups he described. These methods promise to simplify the production of protein microarrays and enable further applications.

In the final session, a protein enrichment method called Global Proteome Survey (GPS), in which binders are used to concentrate proteins with short peptide motifs in common, was introduced by Carl Borrebaeck (Lund). This is somewhat akin to the SISCAPA method and will be useful as an adjunct to mass spectometry, where it will improve the detection of low level protein biomarkers. Mike Romanos (Cambridge), CEO of the recently established Crescendo Biologics, then described their combination of transgenic mice making human single VH domain fragments and in vitro ribosome display which will hopefully generate a new generation of therapeutics. The last speaker was John McCafferty (Cambridge), the pioneer of antibody phage display. He described chain shuffling and phage display selections to produce antibody fragments of improved affinity for use in immunoprecipitation and demonstrated applicability to characterisation of a protein network in combination with mass spectrometry. Given that phage display technology has recently passed 20 years since the first publication with John as lead author, this was a fitting conclusion to the meeting.

Assessment of the results & impact of the event on the future direction of the field

This highly successful and enjoyable workshop brought together many of the leading European and US academic and industrial players in affinity proteomics, encouraging personal interactions and networking. Participant feedback indicated that this series of workshops is indeed now one of the best meetings in the field. The meeting highlighted recent progress in affinity proteomics by linking novel developments in proteome analysis, particularly the key area of signal transduction, with the growing interest in personalised medicine and biomarker discovery and detection. These will be of major importance for many years to come. The meeting also promoted the linkage between EU projects such as AFFINOMICS and AffinityProteome with parallel activities in the USA, and was attended by representatives from the NIH and NCI responsible for the Protein Capture Reagents and Clinical Proteomics for Cancer initiatives respectively. A number of papers by presenters at the meeting will be published in a special issue of the journal New Biotechnology.

Programme
Monday 14th March
17:00-17:15 Welcome and introduction
17:15-17:45 Mathias Uhlén
17:45-18:15 Pierre Legrain
18:15-18:30 General Discussion
19:00 Drinks and dinner at Alpbacherhof

Tuesday 15th March
08:45-09:15 Gianni Cesareni
09:15-09:45 Lukas Huber
09:45-10:15 Klaus Lindpaintner
10:15-10:45 Short talks from abstracts:
10:15-10:25 Anke Prinz
10:25-10:35 Saïd Taouji
10:35-10:45 Mariliza Derveni
10:45-11:00 Discussion
11:00-11:30 Coffee break & poster viewing
16:00-16:30 Coffee break & poster viewing
16:30-17:00 Olli Kallioniemi
17:00-17:30 Andreas Plϋckthun
17:30-18:00 Larry Gold
18:00-18:30 Andrew Bradbury
18:30-18:45 Discussion
18:45 Departure from conference centre to walk to Rossmoos
19:30 Dinner at Rossmoos

Wednesday 16th March
08:45-09:15 Bernhard Kuster
09:15-09:45 Ulf Landegren
09:45-10:15 Walter Schubert
10:15-10:45 Short talks from abstracts:
10:15-1025 Fridjtof Lund-Johansen
10:25-10:35 Christoph Schroeder
10:35-10:45 Jochen Schwenk
10:45-11:00 Discussion
11:00-11:30 Coffee break & poster viewing
16:00-16:30 Coffee break & poster viewing
16:30-16:40 Mike Taussig
16:40-17:10 Doron Gerber
17:10-17:30 Carl Borrebaeck
17:30-18:00 Mike Romanos
18:00-18:30 John McCafferty
18:30-18:45 Discussion
19:00 Dinner & party