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3 rd Spanish Human Proteome Project ( spHPP ) Workshop

3 rd Spanish Human Proteome Project ( spHPP ) Workshop. La Cristalera , Miraflores de la Sierra, Madrid, December 10-11th , 2012 Juan P Albar , ProteoRed -ISCIII, CNB-CSIC. The Human Proteome Project Launch. Sydney HUPO2010 23 September 2010 Geneva HUPO2011 12 September 2011.

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3 rd Spanish Human Proteome Project ( spHPP ) Workshop

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  1. 3rdSpanish Human Proteome Project (spHPP) Workshop La Cristalera, Miraflores de la Sierra, Madrid, December 10-11th, 2012 Juan P Albar, ProteoRed-ISCIII, CNB-CSIC

  2. The Human Proteome Project Launch Sydney HUPO2010 23 September 2010 Geneva HUPO2011 12 September 2011 Sydney September 23 Human Proteome Project

  3. Gil’scommentsontheResourcesIssues: A goodexample of a resourceweshouldmakeknowntoall C-HPP and B/D-HPP investigatorsistheChr 16 team'slibrary of 11,000 expressionvectorsfor SRM assays (www.dnasu.org,www.plasmid.med.harvard.edu) as stated in theChr 16 manuscript. I thinkweshouldcollectsuchoffersfromthemanuscripts of theSpecialIssue of JPR and makethemknownwithin HPP. Wecouldalsotabulatethem in theOverviewarticle. Thisinformationcouldmake a goodarticle in HUPOST, ifwehavethecapacitytomakeinformation and resourcesavailable more widely. In addition, theChr 16 teamhighlightedtheirinterest in the B/D-HPP; weneedtothinkabouthowbestto capture and stimulatethechromosometeamstocontributetheirdatasets and possiblytheirspecimenstointerestedcolleaguesthroughoutthe B/D-HPP. Theyidentifyprojects in cancers, obesity, infectious, neurodegenerative, cardiovascular, and rheumatoid and osteoarthritisdiseasecategories. Theleader/correspondingauthoris Fernando Corrales, Pamplona, fjcorrales@unav.es. DatasetSubmissionGuidelinesforthe Human ProteomeProject Version1.0 (2012 October 30): Basedondiscussions at the HUPO ProteomicsStandardsInitiative (PSI) workshop (2012 Mar 12-14), theProteomeXchangeworkshop (2012 Mar 15-16), theWorld HUPO Congress in Boston (2012 Sept 9-13), follow-on emails, and review and approvalbythe HPP-EC. Gel images Gel imagesaccompanying MS/MS datasetsshould be uploaded as supportingraw data withthe MS/MS dataset. Thereis a possibilitythat Swiss2DPage orProteoRedmay be abletoserve as a Gel imagerepository, butthisisnotyetconfirmed.

  4. MICINN ISCIII SteeringCommittee Analytical Gene/chromosomecentric Research Diseasecentric Bioinformatics MS andAbbasedquantitativemethods Open accessrepositories Hallmarksofdisease Potentialbiomarkersandtherapeutic targets Biotechs MICINN Designofprototypes Quantitationoftargetproteins CNB-CSIC PCM-UCM CIB-CSIC FJD/HNP CIC bioGUNE CIMA UAB-CSIC UV PCB VHIO USAL UPV-EHU INIBIC BioDo HM Pharmas ISCIII Clinicaldevices Personalized medicine

  5. CHROMOSOME 16 at PeptideAtlas SITE

  6. 16 Chromosome 16 general aims 16 Bioinformaticanalysis of chromosome 16. Celltype(s) selection. Trascriptomic and proteomicdetailedanalysis. Expression, purification and characterization of proteinsencodedbychromosome 16 thathavenotbeendescribed so far. Development of MRM methodsforthequantification of themostabundantproteinspeciesencodedbyeach of thechromosome 16 genes. Identification, characterization and quantification of proteinvariantsfromchromosome 16 genes. Definition of SOP Development of bioinformaticenvironment. Biobanking. Configuration of the B-D pilar fortheSpanish HPP consortium.

  7. 16 Chromosome 16 workplan 16 2012 2013 2014 • Bioinformatic study • MRM for 184 proteins • MRM for 339 proteins • MRM for 339 proteins • SOP, bioinformatic procedures, formats, data banks, etc. • Validation of MRM assays (3 groups, immunoreagents, correlation with gene expression) • Clinical samples. Disease-related changes on protein abundance. Biomarkers • Biobanking 2015 2016 2017 • Validation of MRM assays (3 groups, immunoreagents, correlation with gene expression) • Clinical samples. Disease-related changes on protein abundance. Biomarkers • Biobanking

  8. Workflow Scheme Chromosome 16 486 HPA antibodiesfor Chr16 proteins, 121forunknown ENSEMBL v68 UniProtKBv2012_06 GPMDB v101012 HPA v10.0 110 OMIM hits Obesity Neurodegenerativediseases Cancer 2316genes 870proteincodinggenes 305unknownproteins Proteinexpressionvectorsfor58 Chr16 unknownproteins Coverage of 75% gene products in Lymphoidcells Epitelial cells Fibroblasts Shotgun Proteomic analysis Transcriptomic analysis Data integration Gene expressionprofile Proteinprofile Global and celltypespecificoutcomes Correlationproteome/transcriptome Proteomecoverage and chromosomedistribution Chr16 proteomecoverage

  9. 16 Workplanproposal 16 • Characterize the transcriptome to define the actual chr16’s gene set expressed and most importantly which genes are not expressed in the three selected cell types. • Detailed characterization of the proteome: Shotgun approaches for maximum protein coverage. • Expression, purification, and characterization of 22 unknown proteins available in NAPPA collection. • Development of SRM quantitative assays. • Protein genes (870) has been distributed in known (565) and unknown groups (305) and the study will be performed along 3 years, starting in 2012 (214 proteins). • Protein packs has been assigned to each of the 8 MRM groups (24), including equal proportion of known (20, with different range of theoretical difficulty) and unknown proteins (4). • Unknown proteins have been first searched by targeted discovery (TD) and relevant information resulting from these studies will be used to design MRM assays. TD groups (9) will analyze 54 proteins during the first year. • Each group will use the master sample. TD will be done in all three selected cell lines. Decision on using a pool or a particular cell line for the unknown proteins will be taken in light of the results from TD. • Validated by 2 additional groups. • Definition of Bioinformatic standards and SOPs. • Definition of Biobanking strategy, standards... in collaboration with the Spanish Biobanking Platform.

  10. 16 Workplanproposalfor 2012 16 • As lymphocytes express more than 75% Chr16 protein coding genes, periferal blood lymphocyte (PBL), or lymphoid cell lines are proposed. Epithelial cell lines and/or fibroblasts might complement. • Once decided the biological source: • Prepare a master sample to distribute to all participating labs (CIMA, CIB, VHIO, others). • Characterize the transcriptome to define the actual chr16’s gen set expressed and most importantly which genes are not expressed (CIMA). • Characterize the proteome in detail (protein sequencing teams). It may require fractionation. • Expression, purification, and characterization of 22 unknown proteins available in NAPPA collection (CIC, sequencing team...).

  11. Development of bioinformatic environment: Spanish HPP will join the different resources to provide a unique enquiring system NProt PRIDE PAtlas External repositories Data standards submission (ProteomeXchange) Proteomics results + + Emsembl Upcoming UProt GPMDB

  12. ProteoRed MIAPE web repository MIAPE MS MIAPE MSI • MIAPE Comparison Project • MIAPE Extractor Software

  13. ProteoRed MIAPE web repository MIAPE MS MIAPE MSI Metadata PRIDE XML

  14. Profiling the Chromosome 16 by high-resolution data-dependent Mass Spectrometry

  15. CHROMOSOME-16 CONSORTIUM STRUCTURE Board of Directors: Principal investigators (PIs) Council Project Manager ExecutiveCommittee 1st Deputy Director 2ndDeputy Director WG 1 WG n . . . . . . . . . . . . . . . . . WorkingGroups ResearchUnits RU 1a RU 1x RU na RU nx . . . . . . . . 3rdSpHPPWorkshop, La Cristalera, Dec 2012

  16. WG 1.-Protein Expression & Peptide Standard Team RU1c (Peptide Standard Team) JP. Albar CNB-CSIC, ProteoRed-ISCIII, Madrid RU1d (m-RNA ExpressionAnalysisTeam) FJ. Corrales CIMA-UN, ProteoRed-ISCIII, Pamplona RU1a (Protein Micro Array) M. Fuentes CIC/IBMCC (USAL/CSIC), Salamanca RU1b (ProteinExpressionTeam) C. Gil UCM-PCM, ProteoRed-ISCIII Madrid ScientificResearchers: M. González N. Dasilva P. Díez M. Perez de Andrés ScientificResearchers: M. Lombardía ScientificResearchers: L. Monteoliva A. Pitarch E. Marín ScientificResearchers: M. Molina 3rdSpHPPWorkshop, La Cristalera, Dec 2012

  17. WG 2.-S/MRM-Protein Platform Team RU2b FJ. Corrales CIMA-UN ProteoRed-ISCIII, Pamplona RU2a JP. Albar CNB-CSIC, ProteoRed-ISCIII, Madrid RU2c F. Canals V.H.I.O. ProteoRed-ISCIII, Barcelona RU2d C. Gil PCM-UCM ProteoRed-ISCIII, Madrid RU2e M. Sanchez del Pino UV ProteoRed-ISCIII, Valencia RU2f F. Vivanco / ME. Barderas FJD,Madrid/HNP, Toledo RU2g FJ. Blanco INIBIC, ProteoRed-ISCIII, La Coruña RU2h S. Barceló IACS, ProteoRed-ISCIII, Zaragoza (Provisional) ScientificResearchers: I. Orera M. Strunk • ScientificResearchers: • M. Marcilla • S. Gharbi • Alpízar • C. González ScientificResearchers:MI. Mora L. Odriozola C. Miqueo R. Martínez ScientificResearchers: ScientificResearchers: N. Colomé JJ.Bech-Serra ScientificResearchers: ML. Hernáez F. Clemente D. Cáceres ScientificResearchers: L. Valero O. Antúnez R.Argamasilla L. Cantero ScientificResearchers: G. Alvarez M. Posada M. Martín L. González F. de la Cuesta L. Mouriño V. Moral G. Barroso LR. Padial ScientificResearchers: P. Fernandez V. Calamia C. Ruiz C. Fernandez Scientific Researchers: 3rdSpHPPWorkshop, La Cristalera, Dec 2012

  18. WG 3.-Protein Sequencing Team RU3a JM. Mato / F. Elortza CIC-BioGUNE, ProteoRed-ISCIII, Bilbao RU3c M. Sánchez del Pino UV, ProteoRed-ISCIII, Valencia RU3d E. de Oliveira PCB, ProteoRed-ISCIII, Barcelona RU3e I. Casal CIB-CSIC, ProteoRed-ISCIII, Madrid RU3f JM. Arizmendi UPV ProteoRed-ISCIII, Bilbao RU3g JP. Albar CNB-CSIC, ProteoRed-ISCIII, Madrid RU3b J. Abian IIBB-CSIC ProteoRed-ISCIII, Barcelona ScientificResearchers: L. Valero O. Antúnez R. Argamasilla L. Cantero V. Rejas • ScientificResearchers: • Paradela • R. Navajas Scientific Researchers: ScientificResearchers: M. Azkargorta E. Rodriguez I. Iloro I. Escobes ScientificResearchers: M. Carrascal J. Villanueva O. Gallardo ScientificResearchers: A. Odena R. Díaz ScientificResearchers: M. Mendes M.Fernandez Scientific Researchers: ScientificResearchers: A. Fullaondo M. J. Omaetxebarria G. Prieto K. Aloria J. Beaskoetxea Scientific Researchers: 3rdSpHPPWorkshop, La Cristalera, Dec 2012

  19. WG 4.-Bioinformatics Team RU4a JP. Albar CNB-CSIC, ProteoRed-ISCIII, Madrid RU4b FJ. Corrales CIMA-UN ProteoRed-ISCIII, Pamplona RU4c J. Abian IIBB-CSIC ProteoRed-ISCIII, Barcelona RU4d C. Gil PCM-UCM ProteoRed-ISCIII, Madrid RU4e JM. Arizmendi UPV ProteoRed-ISCIII, Bilbao RU4f M. Sánchez del Pino UV, ProteoRed-ISCIII, Valencia • ScientificResearchers: • Medina • S. M-Bartolomé ScientificResearchers: V. Segura E. Guruceaga M. Braga ScientificResearchers: O.Gallardo ScientificResearchers: V. Vialás ScientificResearchers: ScientificResearchers: G. Prieto ScientificResearchers: J. Ortíz 3rdSpHPPWorkshop, La Cristalera, Dec 2012

  20. WG 5.-Clinical Health Care & Biobanking Team (B/D-SpHPP) RU5a FJ. Blanco INIBIC, ProteoRed-ISCIII, La Coruña RU5b F. Bonilla/ I. Casal /C. Belda Hospital Puerta de Hierro, CIB Madrid RU5c L. Rodríguez Padial/ F. Vivanco /M.E Barderas FJD, Madrid/HNP, Toledo RU5d A. López de Munain Inst. Biodonostia S. Sebastian RU5e F. Corrales CIMA-UN ProteoRed-ISCIII, Pamplona RU5f C. Gil PCM-UCM ProteoRed-ISCIII, Madrid RU5g M. Morente SNBN, CNIO, Madrid (considered for inclusion in the consortium) ScientificResearchers: B. Cillero J. Mateo B. Rocha L. Lourido ScientificResearchers: ScientificResearchers: C. Peña S. Torres M. Mendes ScientificResearchers: G. Alvarez M. Posada M. Martín L. González F. de la Cuesta L. Mouriño V. Moral G. Barroso ScientificResearchers: J.M. Arizmendi A. Fullaondo MJ. Omaetxebarria G. Prieto K. Aloria J. Beaskoetxea Scientific Researchers: ScientificResearchers: A. Moreno E. Bigaud ScientificResearchers: L.Monteoliva A.Pitarch E. Marín ScientificResearchers: F. de Luna MC. Marín 3rdSpHPPWorkshop, La Cristalera, Dec 2012

  21. WG1-Protein expression & Peptide Standard Team RU1a (Protein Micro Array and Protein Expression Team):Dr. Manuel Fuentes Centro de Investigación del Cáncer/IBMCC (USAL/CSIC), Salamanca RU1b (Peptide standardization Team): Dr. Juan P Albar National Center for Biotechnology-CSIC (CNB-CSIC), ProteoRed, Madrid WG3-Protein Sequencing Team RU3a-Dr. José M. Mato / Dr. Félix Elortza CIC-BioGUNE, ProteoRed, Bilbao RU3b-Dr. Joaquin Abian IIBB, CSIC ProteoRed, Barcelona RU3c-Dr. Manuel Sanchez del Pino University of Valencia, ProteoRed, Valencia RU3d-Dr. Eliandre de Oliveira ParcCientífic de Barcelona (PCB), ProteoRed, Barcelona RU3e-Dr. Ignacio Casal Biology Research Center-CSIC (CIB-CSIC), ProteoRed, Madrid RU3f-Dr. Jesus M. Arizmendi Basque Country University ProteoRed, Bilbao RU3g-Dr. Juan P Albar National Center for Biotechnology-CSIC (CNB-CSIC), ProteoRed, Madrid WG2-S/MRM-Protein Platform Team RU2a-Dr. Juan P Albar National Center for Biotechnology-CSIC (CNB-CSIC), ProteoRed, Madrid RU2b-Dr. Fernando Corrales CIMA-University of Navarra, ProteoRed, Pamplona RU2c-Dr. Francesc Canals Valld'HebronInstitutd'Oncologia (V.H.I.O.),ProteoRed, Barcelona RU2d-Dr. Concha Gil Madrid Science Park-Computensis University (PCM-UCM), ProteoRed, Madrid RU2e-Dr. Manuel Sanchez del Pino Felipe Princess Research Center (CIPF), ProteoRed, Valencia RU2f-Dr. Fernando Vivanco / Dr. ME Barderas Jimenez Díaz Foundation (FJD), Madrid /National Paraplegics Hospital, Toledo RU2g-Dr. Francisco Blanco Instituto de InvestigacionBiomedica (INIBIC), ProteoRed, La Coruña RU2h-Dr Silvia Barceló Aragón Health Sciences Institute (IACS), Zaragoza RU2i- Dr. Eduard Sabidó Centre de regulacióGenómica (CRG-UPF), ProteoRed, Barcelona WG5-Clinical Health Care & Biobanking Team RU5a-Dr. Francisco Blanco Instituto de InvestigacionBiomedica (INIBIC), ProteoRed, La Coruña RU5b-Dr. Felix Bonilla/ Dr Ignacio Casal Hospital Puerta de Hierro, CIB-CSIC Madrid RU5c-Dr. Rodriguez Padial/Dr. Fernando Vivanco/ Dr. ME Barderas Jimenez Díaz Foundation (FJD), Madrid /National Paraplegics Hospital, Toledo Ru5d-Dr. Adolfo López de Munain InstitutoBiodonostia, San Sebastian RU5e-Dr. Fernando Corrales/J. Prieto CIMA-University of Navarra RU5f-Dr. Concha Gil PCM-Computensis University (PCM-UCM), ProteoRed, Madrid RU5g-Dr. Manuel Morente(considered for inclusion in the consortium), Spanish National BioBank Network- ISCIII. CNIO, Madrid” WG4-Bioinformatics Team RU4a-Dr. Juan P Albar National Center for Biotechnology-CSIC (CNB-CSIC), ProteoRed, Madrid RU4b-Dr. Fernando Corrales CIMA-University of Navarra ProteoRed, Pamplona RU4c-Dr. Joaquin Abian IIBB, CSIC, ProteoRed, Barcelona RU4d-Dr. Concha Gil PCM-Computensis University (PCM-UCM), ProteoRed, Madrid RU4e-Dr. Jesus M. Arizmendi Basque Country University, ProteoRed, Bilbao RU4d-Dr. Manuel Sanchez del Pino University of Valencia, ProteoRed, Valencia 3rdSpHPPWorkshop, La Cristalera, Dec 2012

  22. 2013 SpHPP HALLMARKS • 1. European Bioinformatics cross analysis of Jurkat cells LC-MS/MS data. • 2. PM8/QTAPAS Targeted Proteomics Study. • 3. Specific WGs Hallmarks: • CHPP: Berlin, March 16th, 2013 • HUPO-PSI-PX: Liverpool, April 15th-19th, 2013 • HUPO: Yokohama, September 2013 - 2013 CHPP Report, December 2013 SpHPPCouncil, La Cristalera, Dec 2012

  23. III Workshop of theChromosome 16 ConsortiumAgenda • 10-11 Dic. 2012, La Cristalera, Miraflores de la Sierra, Madrid • 10 de Diciembre (1): • 10:30 Bus CNB-Miraflores de la Sierra • 11:30-11:45 Bienvenida Juan P. Albar • 11:45-13:30 WG 1 Expresión de proteínas. Síntesis de Péptidos • Introducción. Concha Gil. (5´) • Síntesis de péptidos. Manuel Lombardía. (15´) • Expresión de Proteínas. Estrategia general. Manuel Fuentes. (20´) • Análisis de las proteínas expresadas mediante MS. Félix Elortza. (15´) • Otras estrategias de expresión. Felipe Clemente y Elvira Marín. (15´) • Discusión y participación de otros grupos en el WG1. 15 min • Plan de trabajo Junio 2013. Juan Pablo Albar, Concha Gil, Manuel Fuentes. (20´) • 13:30-13:45 WG5 Biology and Disease (B/D-HPP) • Presentación general. (15´) • 13:45-15:15 Comida 3rdSpHPPWorkshop, La Cristalera, Dec 2012

  24. 15:15-16:15Consejo de dirección: Reunión de los investigadores principales (IPs) • Gestión del proyecto. Posibles escenarios en función de la financiación • Hitos para el 2013. Relación C-HPP con B/D-HPP • 16:15-19:30WG 3 ShotgunproteomicsGroup • Presentación general. (30’) • Fernando Corrales: Comparación de resultados de shotgun y transcriptómica. (15’) • SeverineGharbi/Carmen González: ProfilingtheChromosome 16 byhigh-resolutiondatadependent MS: Extraction/fractionationmethodevaluation in Jurkatcells. (15´) • Óscar Gallardo/Joan Villanueva: Experimentos de shotgun empleando listas de inclusión. Utilización de datos de SEQUEST con MIAPE Extractor. (15’) • 17:30-18:00 Coffee break • KermanAloria. ProteinGrouping. (15’) • Alberto Medina. How and wherewill I applythe FDR filter? (10') • Discusión: (50’) • Estrategias para ampliar la cobertura del cromosoma 16. Utilización de la información de los experimentos shot-gun para el diseño de experimentos de MRM.Información cuantitativa de los datos de shot-gun • Plan de trabajo Junio 2013. (15’) • 19:30-20:15 Charla Workshop Speaker Invitado: • Juan Antonio Vizcaíno: “PRIDE and theProteomeXchangeconsortium: enhancingvisibility and accessibility of MS-basedproteomics data”. (30’) • Discusión. (15’) 3rdSpHPPWorkshop, La Cristalera, Dec 2012

  25. 11 de Diciembre (1): • 9:00-11:15WG2 SelectedReactionMonitoringGroup (S/MRM) • Presentación general (30´) • MRM data base. Vital Vialas (15'). • Discusión (60’) • Aspectos a discutir: • - Criterios verificación transiciones- péptidos • - Validación con péptidos sintéticos: criterios para selección de péptidos • - Criterios de "proteotipicidad" - Tratamiento de las isoformas • - Factores para establecer un sistema  de scores- ranking de péptidos para • cuantificar por SRM (presencia de Met, Cys, modificaciones...) • - Optimización de parámetros de adquisición: Energías de Colisión... • - Uso de standards para determinar tiempos de retención relativos • - Uso de un standard interno para establecer una escala de intensidad • relativa de las señales de SRM • - Planificación validación multi-laboratorio • -Objetivos siguiente hito: validación, asignación de nuevas proteínas... • Plan de trabajo Junio 2013 (15’). • 11:15-11:45Café 3rdSpHPPWorkshop, La Cristalera, Dec 2012

  26. 11 de Diciembre (2): • 11:45-13:30WG4 BioinformaticsGroup • Presentación general: Bioinformatics pipeline improvements. Alberto Medina (30’) • Discusión (60') • Propuestas Logo/Imagen. • Actualización BDsReferncia • Problemas envío datos MIAPE extractor • Consultas datos shotgun para diseño experimentos MRM. • Plan de trabajo Junio 2013 (15’). • 13:30-14:45 Conclusiones finales. • 15:00-16:00 Comida • 16:30 Bus Miraflores-CNB 3rdSpHPPWorkshop, La Cristalera, Dec 2012

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