Relevance of Proteomics Standards for ProteoRed. BIOINFORMATICS ANALYSIS FROM PROTEOMICS DATA WORKSHOP ProteoRed Bioinformatics Workshop Salamanca, March 2009 Juan Pablo Albar ProteoRed General Coordinator

¿ WHAT IS ProteoRed? Spanish network for the coordination, integration and development of proteomics facilities The network is coordinated from the National Center for Biotechnology in Madrid, asuming operational and scientific responsabilities

Node 2 J. Mato - CIC-bioGUNE F. Corrales – CIMA JM. Arizmendi - UPV Central Structure JP. Albar - CNB J. Mato - CIC-bioGUNE J. Abian - LP-CSIC/UAB Assoc. to Node 1 I. Casal - CIB Assoc. To Node 5 P. Carrasco - UV Node 5 M. Sánchez del Pino – CIPF R. Bru - UA Node6 JA. Bárcena - UCO Node 1 JP. Albar - CNB C. Gil - PCM-UCM A. Marina - CBMSO Node 4 X. Bustelo - CIC Node 3 J. Abian - LP-CSIC/UAB J. Arribas - HUVH S. Martínez - PCB D. Andreu - UPF FX. Avilés - IBB UAB O. Bachs - UB *. * - Assoc. to node 4 F.J. Blanco - INIBIC Portugal Ana Coelho - ITQB Deborah Penque - ProCura Geneva Jean Charles Sanchez - UG Geographical organization

1.1 Million Euros / Year Total Budget: 6.3 M Euros / 5 years period 80% Personnel 3% Traveling Other activities 8% Reagents 9% 0,8 M € Equipment

ProteoRed main objectives are the standardization and quality control of the services offered by their members and thus is a quality label for the users. To reach these aims seven working groups have been created, including one devoted to the “Standardization of protocols and technologies”. ProteoRed activities started on September Main Objectives

WG 1. Technological development and standardization of protocols Alberto Paradela, Juan Pablo Albar, Joaquín Abián,... Functional organization WG 2. Samples collection and handling Francesc Canals, Concha Gil,... WG 3. Bioinformatics support Juan Pablo Albar, Salvador Martínez de Bartolomé,... WG 4. Functional organization of proteomic services and coordination of price scales. Eliandre de Oliveira, Félix Elortza,... WG 5. Training, education and diffusion Concha Gil, Jesús Mª Arizmendi,... WG 6. Internationalization and coordination with other proteomic consortia or international platforms. José Mª Mato and Juan Pablo Albar WG 7. Biomedical Proteomics Francisco Blanco, Juan Pablo Albar, …

Main activities ProteoRed Multicenter Experiments HUPO-PSI standards implementation Organization of training courses Active collaborations Other activities

Main activities ProteoRed Multicenter Experiments HUPO-PSI standards implementation Organization of training courses Active collaborations Other activities

Main activities ProteoRed Multicenter Experiments HUPO-PSI standards implementation A real compromise: Standarization initiatives and ProteoRed

Standards Within ProteoRed Consortium –In-lab workflow normalization (WG1+2) ABRF International study/contest participations Multi-Centric wet experiments –Experiment reporting and reproducibility: automatic generation of MIAPE documents (WG3+4) – Experiment data exchange: PRIDE incorporation into daily pipeline (WG3)

Standards Within ProteoRed Consortium –In-lab workflow normalization (WG1+2) ABRF International study/contest participations Multi-Centric wet experiments –Experiment reporting and reproducibility: automatic generation of MIAPE documents (WG3+4) – Experiment data exchange: PRIDE incorporation into daily pipeline (WG3)

ProteoRed contribution to ABRF sPRG 2006 study: Evaluation of a prototype standard protein mixture (4 th quarter of 2005) ProteoRed WG1 organized the PME1 Study (Sigma-Aldrich UPS 2006 study) : Evaluation of a commercial standard protein mixture (May- October 2006) ProteoRed contribution to ABRF sPRG 2007 study: Evaluation of a prototype standard phosphoprotein mixture (September February 2007) ProteoRed contribution to ABRF PRG 2008 study: Qualitative Proteomics Study – Identifying differences in primary structure (4 th quarter of 2007) ProteoRed contribution to ABRF PRG 2009 study: Relative Protein Quantification in a Clinical Matrix Study (4th quarter of 2008) Proteored contribution to ABRF PRG 2010 study: Determining sample composition and identifying unforeseen problems in otherwise ‘straight-forward’ proteomics analyses (4 th quarter of 2009) WG 1.- Technological development and standardization of protocols

These contests have been useful to evaluate the performance of the Spanish Proteomics labs and to know if they reached international standards. Several different experimental methodologies have been evaluated along these years, including protein identification, phosphoprotein analysis and qualitative proteomics. Some of the contests served to incorporate previously unattended methodologies, e.g., phosphoprotein analysis. The contests were useful to evaluate new software platforms purchased by ProteoRed, e.g., Phenyx and Peaks Search Engines.

Standards Within ProteoRed Consortium –In-lab workflow normalization (WG1+2) ABRF International study/contest participations Multi-Centric wet experiments –Experiment reporting and reproducibility: automatic generation of MIAPE documents (WG3+4) – Experiment data exchange: PRIDE incorporation into daily pipeline (WG3)

2006: 1) 2DE Standard: testing each lab 2DE Protocols 2)PMF Protein Identification capabilities 2007: 2DE Multicentric assay to study reproducibility of different 2DE procedures (Silver, SYPRO, DIGE) 2008: Multicentric study: Reproducibility of a 2D Differential Proteomic Experiment : PME-5: ProteoRed Quantitative Proteomics Study. WG 2.- Samples collection and handling MIAPE GE, GI, MS and MSI compliant documents, generated by ProteoRed, have been used to report and compare results +100 MIAPE reports from ProteoRed multicenter experiments ( ) are publicly available through web application (

1)- Evaluate robustness of a 2D-DIGE workflow on a differential proteomic analysis. 2)- Strict 2DE conditions to allow for Inter-Lab comparison 3) - Comparison of Image Analysis tools 4) – Performance of MS Protein Id. 5) – Comparison with other LC-MS analysis strategies 6) – Continue to test MIAPE doc. generator through web site Multi-laboratory assay 2008 : Reproducibility of a Differential Proteomic Experiment

Summary Good reproducibility within and across lab Good reproducibility of differentially expressed proteins across labs Supports the ability to validate each others “discoveries” Demonstrates that across lab quality control schemes are feasible within routine 2D proteomic facilities Highlights some areas to emphasise adherence to or requirement of further protocol definitions 17

Standards Within ProteoRed Consortium –In-lab workflow normalization (WG1+2) ABRF International study/contest participations Multi-Centric wet experiments –Experiment reporting and reproducibility: automatic generation of MIAPE documents (WG3+4) – Experiment data exchange: PRIDE incorporation into daily pipeline (WG3)

ProteoRed is assuming HUPO STANDARDS. –Provide to our costumers a MIAPE compliant report attached to the results as a new service quality label WG3 (bioinformatics support working group) has developed a web system that helps to create MIAPE documents: MIAPE generator tool Provide a unique standard compliant report Standard reporting in ProteoRed First step:

MIAPE generator tool or

Generate XML Generate MIAPE report Delete MIAPEEdit MIAPE Public link to the report Share document MIAPE generator tool

MIAPE comparator Compare several MIAPE documents (from different experiments), showing previously selected sections. Allows to identify differential results caused by a particular protocol step, technology used, db search parameter…

MIAPE repository You can share your MIAPE reports with other users or get them available for the whole scientific community It can help in the reviewing phase of a journal publication process 1.Authors can report their proteomics experiment following MIAPE guidelines using the tool 2.Authors submit it to the journal a)as part of the Material and Methods section or as Supplementary Information or… b)…share the report with journal reviewers 3.Journal reviewers easily verify MIAPE-compliance of the experiment by logging in the web tool

Standards Within ProteoRed Consortium –In-lab workflow normalization (WG1+2) ABRF International study/contest participations Multi-Centric wet experiments –Experiment reporting and reproducibility: automatic generation of MIAPE documents (WG3+4) –Experiment data exchange: PRIDE incorporation into daily pipeline (WG3)

Bioinformatics tools –Virtual2D ( This tool shows a set of proteins according to a 2d gel. ProteoRed CNB bioinformatics team (A. Medina, S. Martínez- de-Bartolomé, E. Salazar and M.A. López-García) has developed bioinformatics tools in compliance with PRIDE standard:. –Protein Information and Knowledge Extractor (PIKE: This tool retrieves from the Internet, biological information regarding to a set of proteins. It can create a new PRIDE file from Protein ids. –PRIDE viewer ( represents the information from a PRIDE file in a friendly-user manner WG 3.-Bioinformatics support –MIAPE-PRIDE API: Automatic generation of MIAPE-MS and MSI reports from PRIDE documents and vice-versa (in development). –MIAPE to GelML translator: Automatics generation of HUPO-PSI GelML docuemnts from MIAPE-GE reports. Part of GelML specification article (Jones et al. Submitted) MIAPE: SAMPLE PREPARATION PROPOSAL FORMS: MIASPPE Montserrat Carrascal, David Ovelleiro, Marina Gay and Joaquin Abian.

PIKE. Medina et al. (submitted to Proteomics) WG 3.-Bioinformatics support

PRIDEViewer. Medina et al. (in preparation. Proteomics) WG 3.-Bioinformatics support

MIAPE JAVA API WG 3.-Bioinformatics support

Main activities ProteoRed Multicenter Experiments HUPO-PSI standards implementation Organization of training courses Active collaborations Other activities

WG 5.- Education, training and diffusion WGs ovierviews Educational programs of proteomics technologies and applications focusses on: Courses Workshops Laboratory networking Collaboration with: Swiss Bioinformatics Institute (SIB) European Proteomics Association (EuPA) Spanish Proteomics Society (SEProt)

ProteoRed as an example of a collaborative initiative Year 1Year 2Year 3Year 4 Management Meetings WG Meetings Workshops Deliverables External Committees Meetings--13*3*4 TOTAL116 * ISRTC Meeting + Users Committee Meeting + SAB Meeting ProteoRed meetings:

… in summary ProteoRed has established itself as a high level proteomics service provider. ProteoRed is a collaborative network. ProteoRed has consolidated as one of the first institutions really involved in HUPO standards incorporation. The multicentric experiments allows to know the robustness of laboratory workflows as well as the standards implementation.

… in summary ProteoRed is highly active incorporating HUPO PSI standards. ProteoRed has already free accessible tools incorporating HUPO – Standards: MIAPE generator, comparator, repository web tools We are developing new tools PRIDE compilance: PIKE Virtual 2D PRIDE Viewer MIAPE-PRIDE API