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Proteomics facility at the Institute of Molecular Biology and Biotechnology

Final Report Summary - PROFI (Proteomics facility at the Institute of Molecular Biology and Biotechnology)

Executive Summary:

The goal of ProFI was to enhance the research potential of the Institute of Molecular Biology and Biotechnology (IMBB), in the areas of proteomics and systems biology and to provide immediate and sustained support for research excellence to its junior and promising researchers. Collectively, ProFI allowed the IMBB to strengthen its status as a centre of fundamental biological research excellence in the EU periphery. It strengthened research efforts that directly address the amelioration of quality of life (such as research by Profi members on microbial pathogenesis and ageing etc) and it strengthened IMBB's capacity to secure competitive EU and International funding. ProFI accomplished a number of specific objectives within this project:
1: Establishment of a proteomics facility at IMBB ( with he following operational modules: Protein purification and separation, Protein analysis and sample preparation for Mass Spectrometry, Mass spectrometry, Bioinformatics analysis).
2: Recruitment of one experienced research technician and two to three experienced Researchers (one operational in recombinant DNA technology, protein purification and characterization and the in-house large-scale fermentation facility; 2 ER for focus primarily on proteomics-based research).
3: Mentor labs: Twinning with proteomics centres of excellence.
4: Workshops, conferences and dissemination.
5: Establishment of an Advisory/Evaluation committee.

Having an in house functional proteomics facility IMBB allowed us to address a number of pertinent research objectives. Some of these questions have provided pilot projects for the setting up of the ProFI lab, while others have a more long-term scope and span the full breadth of IMBB research activities:
- Understanding protein trafficking: how proteins are correctly targeted to cell membranes and subsequently cross or integrate into them; focus on bacterial protein export pathways and develop membrane proteomics of cancer cells.
- Biogenesis of mitochondria and their relevance in health and disease; Role of the mitochondrial proteome in mediating oxidative folding and signalling.
- Functional characterization of chromosomal interactions of gene loci involved in the regulation of the immune system and identification of the protein complexes that generate and/or maintain the interchromosomal interactions.
- Systems-level understanding of aging using mice and C.elegans as model organisms to delineate key pathways involved in lifespan regulation.
- Study of the molecular mechanisms that manage the passage of the malaria parasite through the mosquito midgut and the further development of Plasmodium within the anopheline vector.
- Research in the field of computational systems biology with a focus on the development and application of in computo modelling techniques for the investigation of protein-protein interactions in the formation of networks in the cell.

The project was succesfuly completed after a s month extention. During the course of the past 3.5 years ProFI has managed to become a centerpiece of IMBB research. It has connected to activities within IMBB, the University hospital, other university of Crete departments (e.g. Chemistry, Materials Science, Informatics etc) and companies (e.g. Fasmatech).

Project Context and Objectives:

The goal of ProFI was to enhance the research potential of the Institute of Molecular Biology and
Biotechnology (IMBB), in the areas of proteomics and systems biology and to provide immediate and sustained support for research excellence to its junior and promising researchers. In retrospect, ProFI has allowed the IMBB to strengthen its status as a centre of fundamental biological research excellence in the EU periphery. It has also strengthened IMBB's capacity to secure additional competitive funding. To achieve these goals we developed five parallel actions that also represent specific objectives within this project:
a. The purchasing of research equipment that will establish a Proteomics facility at IMBB.
b. The recruitment of three experienced researchers and one experienced research technician c. the twinning of IMBB with European leaders in proteomics research (“mentor labs”).
d. the transnational exchanges of research staff to/from the mentor labs
e. The organization of workshops and conferences
f. The establishment of a panel of experts who are leading scientists who will constantly monitor the project's progress and who will also evaluate its success upon completion.

All of these objectives have been fully met. The vast majority of them are now completed or are in advanced stages of maturation and will be detailed below. The main activities that are ongoing and which are central to the success of ProFI are dissemination, workshops and conferences and exchange visits.

An in house functional proteomics facility helped IMBB address a number of pertinent research objectives. Some of these questions have already provided pilot projects for the setting up of the ProFI lab, while others have a more long-term scope:
- Understanding protein trafficking: how proteins are correctly targeted to cell membranes and subsequently cross or integrate into them; focus on bacterial protein export pathways and develop membrane proteomics of cancer cells.
- Biogenesis of mitochondria and their relevance in health and disease; Role of the mitochondrial proteome in mediating oxidative folding and signalling.
- Functional characterization of chromosomal interactions of gene loci involved in the regulation of the immune system and identification of the protein complexes that generate and/or maintain the inter-chromosomal interactions.
- Systems-level understanding of aging using mice and C.elegans as model organisms to delineate key pathways involved in lifespan regulation.
- Study of the molecular mechanisms that manage the passage of the malaria parasite through the mosquito midgut and the further development of Plasmodium within the anopheline vector.
- Research in the field of computational systems biology with a focus on the development and application of in computo modelling techniques for the investigation of protein-protein interactions in the formation of networks in the cell.

Operational benefits:
a. Retaining the Institute’s existing excellence in basic biology of single proteins or systems with a handful of proteins;
b. Expanding to essential but more "costly" system-level dissection of proteins in living organisms;
c. Open new vistas for possible use of the basic knowledge amassed to improve the quality of life;
d. Secure future excellence by constantly bringing in new talent;
e. Attract necessary international funding for all of the above to become possible
Strengthening of the IMBBs research potential of proteomics and systems biology. Also close contacts that were created as aprt of ProFI meetings and the first Greek Orbitrap users workshop suggested that this benefit will be transferred to other labs in Greece and hopefully to the European Science Area as a whole. IMBB has enhanced its visibility and has attracted necessary national and international funding due to ProFI and since its start of more than 550K Euros in fresh grant support.

Academic Excellence:
ProFI has contributed towards securing future excellence by bringing in new research talent. Profi was the specific reason of attracting 2 junior PIs and 2 postdocs, while ProFI-based research has led to 8 publications in journals and international meetings.

Research and society:
IMBB aims to retain the Institute’s existing edge in basic biology, strengthened by input from ProFI to understand and combat Cancer, Neurodegenerative and infectious disease and provide insight in Stem cells. This involves the development of modern molecular diagnostics, biomarker discovery and novel drugs.

Project Results:

Introduction
The goal of ProFI was to enhance the research potential of the Institute of Molecular Biology and Biotechnology (IMBB), in the areas of proteomics and systems biology and to provide immediate and sustained support for research excellence to its junior and promising researchers. In retrospect, ProFI has allowed the IMBB to strengthen its status as a centre of fundamental biological research excellence in the EU periphery. It has also strengthened IMBB's capacity to secure additional competitive funding from calls that were simply unapproachable a while back.

Progress report on Profi
IMBB is involved in addressing a number of biological questions. Over these years the IMBB has established a strong base in a number of biological disciplines, most prominently in Drosophila and mosquito molecular genetics and genomics, in gene expression mechanisms and in development. Biology is now moving to a post-genomic era, with a demand for so-called "functional genomics" studies where research questions focus on the proteins of an organism, how they interact, how that move, how they catalyze life's chemistries and on how all of these activities provide the properties of the whole biological "system". ProFI was designed as a project that would bring whole organism –omics analysis to the IMBB while on the other hand allowing the IMBB to strengthen its status as a centre of fundamental biological research excellence in the EU periphery. The proteomics aspect to IMBB's research was envisage to be coupled to some significant investment in DNA and RNA-based omics work. To achieve these goals and in compliance with the plan set forth in the original application ProFI explored five parallel actions:

Action 1: Establishment of a proteomics facility at IMBB (ProFI).
This action dealt with purchasing and setting up equipment and preparing an appropriate lab space to house these activities so that proteomics studies can be performed at the IMBB. The necessary instruments in ProFI comprise four modules necessary to secure the flow from organism to data to knowledge (Fig. 1):

Module A. Protein purification and separation
1. Gel-based 2-dimensional protein separation in spots
2. Liquid chromatography
3. Preparative Isoelectric Focusing
Module B. Protein analysis and sample preparation for Mass Spectrometry
1. Visualization and spot marking
2. Protein spot picking
3. Protein removal from gel and digestion
Module C. Mass spectrometry
Module D. Bioinformatics analysis

The list of equipment that was purchased for ProFI was finalized with the help of the Mentors who have been actively engaged in offering advice and suggestions on designing an optimal workflow for ProFI. Following the mentors' advice decisions on the equipment line-up followed a reverse flow prioritization, i.e. the best possible Mass Spectrometry and bioinformatics solution (Modules C and D) were secured first since these are the most critical instruments for an optimal proteomics solution. Then the contents of module A and B were finalized. To ensure we can start the project on day one and optimize the length of depreciation time that is covered by our budget we did extensive background work prior to the start of the grant. This included all the market research for the main equipment, preparatory infrastructure work for the Lab and finalizing all the necessary paperwork for the international tender. As a result we were in a position to move speedily through all the subsequent steps and have the first instruments installed within weeks of the grants start and all testing of the core LC-MS systems including most of the training of the users completed by May 15 of 2009 (see attached timeline figure).

Under the close scrutiny of the hired experienced research technician (ERT), the purchased instruments that comprise the four modules of the facility were installed in appropriately prepared premises at the IMBB in Iraklio. Planning for the appropriate infrastructure was executed together with the suppliers so that the recommended installation features were optimal. The ERT organized the installation visits of the company technicians and interfaced with the IMBB electronics, maintenance and computing support personnel to ensure that all technical, electrical, telecommunications requirements necessary for installation of the instruments were met. The ERT informed the co-ordinator and in the weekly ProFI meetings the management committee of the progress with the installation. ProFI occupies space in the StepC building of FoRTH totalling 67 m.2 This includes a main wet lab and bioinformatics analysis area, a Mass spectrometry room and auxhilliary lab space with controlled temperature rooms and a storage space (See attached figure).
The list of equipment that was purchased for ProFI was finalized with the help of the Mentors who have been actively engaged in offering advice and suggestions on designing an optimal workflow for ProFI. Following the mentors' advice decisions on the equipment line-up followed a reverse flow prioritization, i.e. the best possible Mass Spectrometry and bioinformatics solution (Modules C and D) were secured first since these are the most critical instruments for an optimal proteomics solution. Then the contents of module A and B were finalized. To ensure we can start the project on day one and optimize the length of depreciation time that is covered by our budget we did extensive background work prior to the start of the grant. This included all the market research for the main equipment, preparatory infrastructure work for the Lab and finalizing all the necessary paperwork for the international tender. As a result we were in a position to move speedily through all the subsequent steps and have the first instruments installed within weeks of the grants start and all testing of the core LC-MS systems including most of the training of the users completed by May 15 2009 (see attached timeline figure).

Under the close scrutiny of the hired experienced research technician (ERT), the purchased instruments that comprise the four modules of the facility were installed in appropriately prepared premises at the IMBB in Iraklio. Planning for the appropriate infrastructure was executed together with the suppliers so that the recommended installation features were optimal. The ERT organized the installation visits of the company technicians and interfaced with the IMBB electronics, maintenance and computing support personnel to ensure that all technical, electrical, telecommunications requirements necessary for installation of the instruments were met. The ERT informed the co-ordinator and in the weekly ProFI meetings the management committee of the progress with the installation. ProFI occupies space in the StepC building of FoRTH totalling 67 m.2 This includes a main wet lab and bioinformatics analysis area, a Mass spectrometry room and auxhilliary lab space with controlled temperature rooms and a storage space (See attached figure).

As soon as instruments were installed the ERT in close collaboration with the experienced researchers (ER) and, where necessary, with the help of other experienced IMBB research technicians, the instruments are tested initially by running control experiments and calibration materials subsequently, upon satisfactory results, with real samples. All such tests have been completed and the first actual results from all of the new instruments of the facility have started becoming available within a month of the mass spectrometer becoming fully operational. The ERT and the ERs in collaboration with scientists in the participating biology labs have presented actual research data of a fully functional facility in D1.4 but also in front of the visiting mentors, at international meetings and in refereed publications (see relevant Deliverables and also updates on the ProFI WWW site: http://profi.imbb.forth.gr/ ). A major deliverable satisfying the goals set out in this effort is D1.7 which demonstrated a flawless functioning of the proteomics facility.

Action 2: Recruitment of one experienced research technician and two to three experienced researchers.
Action 2 objectives were:
a. the efficient transfer of know-how for obtaining, analyzing and interpreting data derived from proteomics studies including advanced biocomputing methods.
b. addressing central biological problems relating to basic cellular mechanisms
c. Train IMBB researchers who are not exposed to proteomics.

Establishing ProFI required hiring experienced and specialized personnel to run it, manage it, maintain it and use it to its full potential as a research tool. Due to the complexity of the equipment and the need for establishment of defined experimental/handling flows we considered the hiring of a skilled experienced research technician (ERT) as essential for the success of the undertaking. The ERT who was hired has a strong analytical chemistry background and experience with liquid chromatography and spectroscopic techniques and use of relevant software. The ERT was further trained by the ERs and the IMBB labs and visited a mentor labs to expand his knowledge. Performance met IMBB standards and the ERT was offered a tenured position as the facility manager. Two experienced researchers (ERs), both with 2-5 years of post-doc experience, were also hired. The 2ERs have been trained in gel-based or liquid chromatography-based proteomics and in mass spectroscopy and bioinformatics analysis of proteomic datasets in recognized leading centres in the EU (university of Frankfurt, Max-Planck Institute for Biochemistry) and USA (Northeastern University). They focus exclusively on proteomics-based research, IMBB user training, participation in seminars and through direct interfacing with IMBB researchers we expect them to render the IMBB researcher community more aware of the power and value of proteomics tools. Thematically, the work that has been carried out covers two of the FP7 Health programme topics: a. Translating research for human health with three sub-topics (Integration of biological data and processes; Translational research in infectious and in major diseases) and b. Biotechnology, generic tools and medical technologies for human health (High-throughput research). The research of the ERs has been extremely successful and were therefore offered tenured positions as IMBB staff scientists.

Action 3: Mentor labs: Twinning with proteomics centers of excellence.
To successfully establish ProFI we sought the assistance of established EU researchers. To this end we have established a twinning network with five european expert labs located in Germany, the UK and Switzerland:
ETH-Institute of Molecular Systems Biology, Prof Ruedi Aebersold (CH)
http://www.imsb.ethz.ch/researchgroup/rudolfa
e-mail: rudolf.aebersold@imsb.biol.ethz.ch

University of Würzburg, Rudolf-Virchow-Center, DFG Research Center for Experimental Biomedicine, Würzburg, Germany, Prof Albert Sickmann (D)
http://www.protein-ms.de/
e-mail: sickmann@isas.de

University of Edinburgh, Wellcome Trust Centre for Cell Biology, Dr Juri Rappsilber (UK)
http://www.wcb.ed.ac.uk/rappsilber.htm
e-mail: juri.rappsilber@ed.ac.uk

Johann Wolfgang Goethe-Universität Frankfurt am Main, Institute of Biochemistry, Biocenter Maxvon, Frankfurt/Germany Prof Robert Tampe (D)
http://www.biochem.unifrankfurt.de/tampe/index.html
e-mail: tampe@em.uni-frankfurt.de

Prof Simon Gaskell (UK, Manchester University, Michael Barber Centre for Mass Spectrometry,) was also an original member of the Mentors group but since he relocated to London and took up a high level administrative position he could not participate in ProFI activities and was therefore replaced by Prof Bruno Domon (Lu) (Centre de Recherche Public de la Santé, Luxembourg Clinical Proteomics) http://www.crp-sante.lu/Competence-centers/Luxembourg-Biomedical-Research-Resources/Luxembourg-Clinical-Proteomics
e-mail: bdomon@crp-sante.lu

In addition and following the mentors advice we extended such collaborating labs to include those of Prof T. Jorgensen (Dennmark) who is an expert in deuterium exchange studies, Prof. Lennard Martens who is a global leader in developing bioinformatics tools for proteomics applications and Prof. Chris Gevaert who pioneered a specialized proteomics technique of wide use (Cofradic). The five mentor labs have provide hands-on training for the IMBB ERT and ERs, have visited Crete to give lecturesed and direct demonstration on the use of equipment at ProFDI and advice on protocols and work-flows. Moreover, they actively participate in the advisory/evaluation committee (AEC).

Action 4: Workshops, conferences and dissemination
As part of ProFI we were involved in the organization of 2 advanced training workshops that will offer hands-on training to IMBB researchers who have little or no prior experience with handling proteins or proteomics. In addition, we organized one international conference.
Action 4 objectives that were the widespread dissemination of ProFI activties and the exposure of research scientists to the research potential of FORTH and the possibility for them to materialize future research within FORTH, were materialized.

Action 5: Establishment of an Advisory/Evaluation committee
To ensure optimal, properly directed efforts in the establishment, integration and managing of ProFI we made use of an Evaluation committee formed by five senior members of the Mentor Labs and the coordinator of ProFI. This committee was put in place early on and succeded in its goals of a. ensuring that the proposal is executed properly throughout its course with strict adherence to the highest standards of excellence. b. To critically assess the improvement in research potential of the IMBB brought about by this project after its completion.


Project expected Impacts and benefit to the IMBB:

Operational benefits:
Strengthening of the IMBBs research potential of proteomics and systems biology. Also close contacts that were created as aprt of ProFI meetings and the first Greek orbitrap users workshop suggested that this benefit will be transferred to other labs in Greece and hopefully to the European Science Area as a whole. IMBB has enhanced its visibility and has attracted necessary national and international funding due to ProFI in the past year of more than 1,550K Euros in fresh grant support.

Academic Excellence:
ProFI has contributed towards securing future excellence by bringing in new research talent. Profi was the specific reason of attracting 2 junior PIs and 2 postdocs, while ProFI-based research has led to 14 publications in journals and international meetings.

Research and society:
IMBB aims to retain the Institute’s existing edge in basic biology, strengthened by input from ProFI to understand and combat Cancer, Neurodegenerative and infectious disease. This involves molecular diagnostics and novel drugs.

Conclusion and future
ProFI the project has progressing smoothly and on schedule. The lab has been set up with state-of-the-art equipment and the experienced researchers to run it have been recruited. Research that is being using the facility has already started and IMBB researchers have taken advantage of it and have presented publications. The mentors have evaluated this effort positively and have been very encouraging in directing ProFI-based research to even more challenging research directions. Following the Mentors’ recommendation we are thinking of mechanisms of better combining the service and the research aspects of the facility. strongly that somehow the “service“ and the „research“ functions are better separated anbd this will allow ProFI to focus on research questions that can make a difference in the global proteomics scence.

Potential Impact:
The scale, breadth and scope of the necessary know-how sought here with ProFI is not available in Greece. It is practically impossible to find the necessary funding for a project of this scale from national funds in a timely manner. Strengthening of the research potential of proteomics and systems biology in Crete will also be beneficial to Greek biological research and to the European Science Area as a whole. The enhanced visibility that IMBB will acquire will also allow it to increase random collisions with researchers in complementary fields of expertise.
Operational benefits:
a. Retaining the Institute’s existing excellence in basic biology of single proteins or systems with a handful of proteins;
b. Expanding to essential but more "costly" system-level dissection of proteins in living organisms;
c. Open new vistas for possible use of the basic knowledge amassed to improve the quality of life;
d. Secure future excellence by constantly bringing in new talent;
e. Attract necessary international funding for all of the above to become possible
Strengthening of the IMBBs research potential of proteomics and systems biology. Also close contacts that were created as aprt of ProFI meetings and the first Greek Orbitrap users workshop suggested that this benefit will be transferred to other labs in Greece and hopefully to the European Science Area as a whole. IMBB has enhanced its visibility and has attracted necessary national and international funding due to ProFI and since its start of more than 550K Euros in fresh grant support.

Academic Excellence:
ProFI has contributed towards securing future excellence by bringing in new research talent. Profi was the specific reason of attracting 2 junior PIs and 2 postdocs, while ProFI-based research has led to 8 publications in journals and international meetings.

Research and society:
IMBB aims to retain the Institute’s existing edge in basic biology, strengthened by input from ProFI to understand and combat Cancer, Neurodegenerative and infectious disease and provide insight in Stem cells. This involves the development of modern molecular diagnostics, biomarker discovery and novel drugs.

List of Websites:

http://profi.imbb.forth.gr
Prof Tassos Economou, IMBB-FoRTH, 71110 Iraklio, Crete, Greece
e-mail: aeconomo@imbb.forth.gr