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5 th Spanish Human Proteome Project ( S pHPP ) Workshop

5 th Spanish Human Proteome Project ( S pHPP ) Workshop. La Cristalera , Miraflores de la Sierra, Madrid, November 5-6th , 2013. SpHPP Council, La Cristalera, Nov 2013. AIMS Chromosome 16 annotation .

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5 th Spanish Human Proteome Project ( S pHPP ) Workshop

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  1. 5thSpanish Human Proteome Project (SpHPP) Workshop La Cristalera, Miraflores de la Sierra, Madrid, November 5-6th, 2013 SpHPPCouncil, La Cristalera, Nov 2013

  2. AIMS • Chromosome 16 annotation. • Selection of 3 celllinesbasedonpublishedtranscriptomicprofilesformaximumchromosome 16 coverage • Transcriptomicanalysis of theselectedcelllines. • Shotgunproteomicanalysis of theselectedcelllines. • Development of SRM methodsfor 200 proteins/year. • Expression of missingproteins and development of SRM methods. 32 researchunitsorganized in 5 WG: WG1. Proteinexpression and purification. Peptides. WG2. Development of quantitative S/MRM assays. WG3. Shotgunproteomics. Molecular profiles and PTMs. WG4. Bioinformatics WG5. Biology and disease. Neurodegenerative, cardiovascular, infectious, cancer, obesity, Rheumaticdisorders.

  3. WG1. Composition WG 1.-Protein Expression, Peptide Standard & Micro Array Team RU1c (Peptide Standard Team) Dr. Juan P Albar CNB-CSIC, ProteoRed, Madrid RU1a (Protein Micro Array and ProteinExpressionTeam) Dr. Manuel Fuentes CIC/IBMCC (USAL/CSIC), Salamanca RU1b (ProteinExpressionTeam 2) Dr. Concha Gil UCM-PCM, Madrid RU1e (Bioinformatics,PTMand celllines) Dr. Félix Elortza CIC-BioGUNE Bilbao RU1d (Peptide Standard Team) Dr. Eliandre Oliveira PCB Barcelona ScientificResearchers: M. González N. Dasilva P. Díez M. Pérez de Andrés Scientific Researchers: M. L. Hernáez F. Clemente ScientificResearchers: M. Lombardía

  4. WG1. Unknown protein definition Missing proteins neXtProt: Organizing Protein Knowledge in the Context of HumanProteome Projects J. Proteome Res. 2013, 12, 293−298 Pascale Gaudet,† Ghislaine Argoud-Puy,† Isabelle Cusin,† Paula Duek,† Olivier Evalet,† Alain Gateau,†Anne Gleizes,† Mario Pereira,† Monique Zahn-Zabal,† Catherine Zwahlen,† Amos Bairoch,†,‡and Lydie Lane*,†,‡† CALIPHO group, SIB-Swiss Institute of Bioinformatics, ‡Department of Human Protein Sciences, Faculty of Medicine, University of Geneva, CMU-1, rue Michel Servet 1211 Geneva 4, Switzerland neXtProt Extends the Coverage of Identified Proteins inthe Human Proteome The primary targets of the C-HPP are the so-called “missingproteins” that have not yet been identified by massspectrometry nor detected by antibodies. An important aspectof the C-HPP work is to define which proteins have alreadybeen characterized, to focus on those that have yet to bedetected. To help C-HPP in this task, neXtProt capturesevidence for the existence of each protein based on criteriaestablished by UniProtKB/Swiss-Prot in 2007. Five levels ofevidence have been defined: (1) evidence at protein level (e.g.,identification by mass spectrometry, detection by antibodies,sequence by Edman degradation, or tridimensional structureresolved), (2) evidence at transcript level (e.g., ESTs or fulllength mRNA), (3) inferred by homology (strong sequencesimilarity to known proteins in related species), (4) predictedand (5) uncertain (e.g., dubious sequences that are likely theproducts of erroneous translations of pseudogenes)

  5. Our Quest for the “Missing Proteins”

  6. Chromosome 16 626 HPA antibodiesfor Chr16 proteins, 95formissing neXtProtv2013-10-10 ENSEMBL v73 UniProtKBv2013_09 HPA v11 120 OMIM hits Obesity Neurodegenerativediseases Cancer 2360 genes 840proteincoding genes 143 missingproteins Proteinexpressionvectorsfor 64 Chr16 missingproteins Coverage of 73% 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

  7. WG1. Activities • Bioinformatic analysis. • Updatethemissingproteins data accordingtoneXtProt. • Definitionof tissuesand celllinesforbiologicalvalidation and subcellularlocalization. • 2. Protein expression. • * Clone isolation. Gene sequencing. • * Expression of proteins in a free cell system. • * Purification of expressed proteins. • 3. Development of SRM methodsforexpressedproteins. • 4. SRM assaysin biologicalmatrices (proteomesorsubproteomes of differenttissuesorcelllines). • 5.Synthesize the appropriate peptides for developing quantitative MRM methods.

  8. NeXtProt DATA

  9. WG1. Unkown proteins clonning workflow Plasmid isolation DNA sequencing Bacterial selection

  10. WG1. Unkown proteins cloning Chromosome 16 143 Unkown proteins (260 before) Cloned in pANT7_cGST WG1 28 new proteins Digest with restriction enzymes Sequencing Mass spectrometry (MRM) Expression (IVTT) and purification (GST)

  11. WG1. Unkown proteins expression List Price 1200 IVTT kit 230 Agarosebeads Sampleprice: 40 euros

  12. WG1. Unkown proteins analysis workflow I Expressed protein digestion Skyline peptide/transition selection MRM assay

  13. WG1. Unkown proteins analysis workflow III MRM data checking and MSMS spectra validation

  14. WG1. Unkown proteins results (11 missing protein from 28 expressed protein) 28 Proteinswith selected peptides(Peptides that matched the specifications to be candidate to use it for quantitation) 22 Proteinswith at least three detected peptides in the MRM experiment 24 Proteins with identified peptides by MASCOT in MIDAS experiment.

  15. WG1. Location of unkown proteins

  16. WG1. Unknownproteins PA: protein array, WB: western blot, IF: immunofluorescency

  17. Working plan for Missing Proteins WP 2013-14 • Definition of the Chr16 missing proteins group. As indicated in neXtProt. • Expression of 60 proteins/year. • Development of SRM assays and test in biological matrices. • Peptide synthesis for quantitative SRM methods. • NAPPA and antibody arrays for B/D. October 2013 • Definition of themissinggroupaccordingtoneXtProt. Available NAPPA and antibodyresources. • Definition of tissue and celllinesforbiologicalvalidation. Nov 2013 • Validation of SRM assaysforpreviouslysynthetisedproteins in biological matrices. • Vector production and optimization (??proteins) • Synthesis of peptidesformissingproteinswith no expression vector. Dec 2013 Jan 2014 • Development of SRM methodsfor new missingproteinsusingtherecombinantformorpeptides • Validation in biological matrices. • List of potentialbiomarkersfrom B/D groups Feb2014 March 2014 • Validation of SRM methods in threeindependentlabs. • Design of arraysfor B/D WP. Biomarkers. April2014 May 2014 • Topicsfordiscussion • Expressiononlyformissingproteins? • Forvalidation, selectthetissue/cell line withhigher gene expression. • Recombinantproteinpurification? • Criteriatoselectpeptidesforsynthesis. • ContactJosuaLabaerif clones are notavailable in Salamanca. Groups CNB UCM-PCM PCB CIC-BIOGUNE CIC-USAL Dec 2014 Othergroupsmightalsoparticipate in definingthebestbiologicalmatrix and also in theinterlaboratoryvalidationprocess.

  18. Working plan for Missing Proteins WP 2013-14 • Definition of the Chr16 missing proteins group. As indicated in neXtProt . • Expression of 60 proteins/year • Development of SRM assays • Test in biological matrices. • Peptide synthesis for quantitative SRM methods. • Antibody and NAPPA arrays for B/D groups (2 year, 2015) (Proyectos Manuel Fuentes, Francisco Blanco, Ignacio Casal, Concha Gil, ………….) October 2013 • Definition of themissinggroupaccordingtoneXtProt. • 28 expressedproteins (11 proteinsstillmissingproteins) • Definitionof tissue and celllinesforbiologicalvalidation (Colaborationwith Banco Nacional • de ADN, Banco líneas celulares) • Validation of SRM assaysforpreviouslysynthetisedproteins in biological matrices (3 labs- • MRM group • Synthesis of peptidesformissingproteinswith no expressionvector • Expression of 53 missingproteins (if clones are available) • Developmentof SRM methodsfor new missingproteinsusingtherecombinantformorpeptides • Validation in biological matrices. • Listof potentialbiomarkersfrom B/D groups Nov 2013 Dec 2013 Jan 2014 • . Feb2014 March 2014 April2014 May 2014 Dec 2014

  19. Working plan for Missing Proteins WP 2013-14 • Definition of the Chr16 missing proteins group. As indicated in neXtProt . • Expression of 60 proteins/year • Development of SRM assays • Test in biological matrices. • Peptide synthesis for quantitative SRM methods. • Antibody and NAPPA arrays for B/D groups (2 year, 2015) (Proyectos Manuel Fuentes, Francisco Blanco, Ignacio Casal, Concha Gil, ………….) • Topicsfordiscussion • Expressiononlyformissingproteins?? • Forvalidation, selectthetissue/cell line withhigher gene expression. • Recombinantproteinpurification? • Criteriatoselectpeptidesforsynthesis. • ContactJosuaLabaerif clones are notavailable in Salamanca (done) Groups CNB UCM-PCM PCB CIC-BIOGUNE CIC-USAL Othergroupsmightalsoparticipate in definingthebestbiologicalmatrix and also in theinterlaboratoryvalidationprocess.

  20. WG1. Unknown proteins peptides

  21. WG1. Unknown proteins peptides

  22. WG1. Unkown proteins results. In detail

  23. WG1. Unkown proteins results. In detail

  24. WG1. Unkown proteins results. In detail

  25. WG1. Unkown proteins results. In detail

  26. WG1. Unkown proteins results. In detail

  27. Strategies to Find Missing Proteins • Unusual tissues and cell types: olfactory epithelium, specific brain regions, testis, placenta, oviduct, lung • Development stage: embryo, fetus • Protein families with high homology: keratins, GPCRs, immunoglobulins, histocompatibility antigens—greater sequence coverage and accuracy, variety of methods • Low/very low abundance, high turnover proteins: transcription factors—greater sensitivity; unfavorable physico-chemical properties: hydrophobic, basic, few tryptic sites • Examine RNA-Seq data for evidence of gene expression and mRNA translation (RNC-mRNA)

  28. 66 celltypes • 48 tissues • Clinicalspecimens

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