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

2. 32 research units organized in 5 WG: WG1. Protein expression and purification. Peptides. WG2. Development of quantitative S/MRM assays. WG3. Shotgun proteomics. Molecular profiles and PTMs. WG4. Bioinformatics WG5. Biology and disease. Neurodegenerative, cardiovascular, infectious, cancer, obesity, Rheumatic disorders. AIMS Chromosome 16 annotation. Selection of 3 cell lines based on published transcriptomic profiles for maximum chromosome 16 coverage Transcriptomic analysis of the selected cell lines. Shotgun proteomic analysis of the selected cell lines. Development of SRM methods for 200 proteins/year. Expression of missing proteins and development of SRM methods.

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

5. Missing proteins neXtProt: Organizing Protein Knowledge in the Context of Human Proteome 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 in the Human Proteome The primary targets of the C-HPP are the so-called “missing proteins” that have not yet been identified by mass spectrometry nor detected by antibodies. An important aspect of the C-HPP work is to define which proteins have already been characterized, to focus on those that have yet to be detected. To help C-HPP in this task, neXtProt captures evidence for the existence of each protein based on criteria established by UniProtKB/Swiss-Prot in 2007. Five levels of evidence have been defined: (1) evidence at protein level (e.g., identification by mass spectrometry, detection by antibodies, sequence by Edman degradation, or tridimensional structure resolved), (2) evidence at transcript level (e.g., ESTs or full length mRNA), (3) inferred by homology (strong sequence similarity to known proteins in related species), (4) predicted and (5) uncertain (e.g., dubious sequences that are likely the products of erroneous translations of pseudogenes) WG1. Unknown protein definition

6. Our Quest for the “Missing Proteins”

7. neXtProt v2013-10-10 ENSEMBL v73 UniProtKB v 2013_09 HPA v11 neXtProt v2013-10-10 ENSEMBL v73 UniProtKB v 2013_09 HPA v11 2360 genes 840 protein coding genes 143 missing proteins 2360 genes 840 protein coding genes 143 missing proteins 120 OMIM hits Obesity Neurodegenerative diseases Cancer 120 OMIM hits Obesity Neurodegenerative diseases Cancer Coverage of 73% gene products in Lymphoid cells Epitelial cells Fibroblasts Coverage of 73% gene products in Lymphoid cells Epitelial cells Fibroblasts Shotgun Proteomic analysis Shotgun Proteomic analysis Transcriptomic analysis Transcriptomic analysis 626 HPA antibodies for Chr16 proteins, 95 for missing Protein profile Gene expression profile Data integration Global and cell type specific outcomes Correlation proteome/transcriptome Proteome coverage and chromosome distribution Chr16 proteome coverage Global and cell type specific outcomes Correlation proteome/transcriptome Proteome coverage and chromosome distribution Chr16 proteome coverage Protein expression vectors for 64 Chr16 missing proteins

8. WG1. Activities 1.Bioinformatic analysis. Update the missing proteins data according to neXtProt. Definition of tissues and cell lines for biological validation and subcellular localization. 2. Protein expression. * Clone isolation. Gene sequencing. * Expression of proteins in a free cell system. * Purification of expressed proteins. 3. Development of SRM methods for expressed proteins. 4. SRM assays in biological matrices (proteomes or subproteomes of different tissues or cell lines). 5. Synthesize the appropriate peptides for developing quantitative MRM methods.

9. Description: Chromosome 16 report Name: nextprot_chromosome_16 Release: 2013-09-26 NeXtProt DATA

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

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

12. WG1. Unkown proteins expression List Price 1200 IVTT kit 230 Agarose beads Sample price: 40 euros

13. WG1. Unkown proteins analysis workflow I Expressed protein digestionSkyline peptide/transition selection MRM assay

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

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

16. WG1. Location of unkown proteins

17. WG1. Unknown proteins PA: protein array, WB: western blot, IF: immunofluorescency

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 and test in biological matrices. Peptide synthesis for quantitative SRM methods. NAPPA and antibody arrays for B/D. Definition of the missing group according to neXtProt. Available NAPPA and antibody resources. Definition of tissue and cell lines for biological validation. October 2013 May 2014 Nov 2013 Dec 2013 April2014 March 2014 Feb2014 Jan 2014 Validation of SRM assays for previously synthetised proteins in biological matrices. Vector production and optimization (??proteins) Synthesis of peptides for missing proteins with no expression vector. Topics for discussion Expression only for missing proteins? For validation, select the tissue/cell line with higher gene expression. Recombinant protein purification? Criteria to select peptides for synthesis. Contact Josua Labaer if clones are not available in Salamanca. Development of SRM methods for new missing proteins using the recombinant form or peptides Validation in biological matrices. List of potential biomarkers from B/D groups Validation of SRM methods in three independent labs. Design of arrays for B/D WP. Biomarkers. Groups CNB UCM-PCM PCB CIC-BIOGUNE CIC-USAL Other groups might also participate in defining the best biological matrix and also in the interlaboratory validation process. 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, ………….) Definition of the missing group according to neXtProt. 28 expressed proteins (11 proteins still missing proteins) Definition of tissue and cell lines for biological validation (Colaboration with Banco Nacional de ADN, Banco líneas celulares) Validation of SRM assays for previously synthetised proteins in biological matrices (3 labs- MRM group Synthesis of peptides for missing proteins with no expression vector Expression of 53 missing proteins (if clones are available) Development of SRM methods for new missing proteins using the recombinant form or peptides Validation in biological matrices. List of potential biomarkers from B/D groups October 2013 May 2014 Nov 2013 Dec 2013 April2014 March 2014 Feb2014 Jan 2014. Dec 2014

20. 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, ………….) Topics for discussion Expression only for missing proteins ?? For validation, select the tissue/cell line with higher gene expression. Recombinant protein purification? Criteria to select peptides for synthesis. Contact Josua Labaer if clones are not available in Salamanca (done) Groups CNB UCM-PCM PCB CIC-BIOGUNE CIC-USAL Other groups might also participate in defining the best biological matrix and also in the interlaboratory validation process.

21. WG1. Unknown proteins peptides

23. WG1. Unkown proteins results. In detail

28. 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)

29. 66 cell types 48 tissues Clinical specimens