1. WP 3.2 Chromosome 16: Protein sequencing, characterization and PTM analysis

4. Working plan for SG WP 2013-14 Massive and high accuracy Identification of proteins in Jurkat, MCF7, CCD18 and Ramos cell lines. Grouping Subproteome analysis, secretome, cell membrane and nucleus. Other cell lines and tissues: Huh7, chondrocytes and brain. Isoform characterization: ATMs, SNPs, PTMs Chr16. Analysis of phosphorylation, glycosylation and acetylation under basal and stimulated conditions. Protocol and data analysis. Tools. New strategies October 2013 May 2014 Nov 2013 Dec 2013 April2014 March 2014 Feb2014 Jan 2014 Incorporation of data from new cell lines/tissues. Isolation of subcellular fractions and enrichment test. Working plan for PTMs. Cells, conditions, groups... SG analysis of subcellular fractions. Sample preparation for PTMs analysis. PTMs MS analysis Data analysis and integration. Groups CNB CBM CIB PCM-UCM EHU/UPV CIC BIOGUNE IBB-CSIC PCB VHIO CRG CIC-USAL UV IDIBELL-IAS CNIO CIMA-NAVBIO Definition of additional cell lines/tissues Protocols for subproteome and proteoforms analysis. Coordinators of the two initiatives and groups involved.

5. WG 3.-Protein Sequencing Team RU3a JM. Mato / F. Elortza CIC-BioGUNE, ProteoRed- ISCIII, Bilbao Chair: Ignacio Casal Co-chair: Manuel Sánchez del Pino RU3b J. Abian IIBB-CSIC ProteoRed- ISCIII, Barcelona 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/EHU ProteoRed- ISCIII, Bilbao RU3g JP. Albar CNB-CSIC, ProteoRed- ISCIII, Madrid RU3h M.Esteller/S. Barceló (PTMs) IDIBELL, Barcelona RU3h M.Esteller/S. Barceló (PTMs) IDIBELL, Barcelona Scientific Researchers: Azkargorta M. Escobes I. Iloro I. Rodríguez E. Scientific Researchers: Carrascal M. Gallardo O. Jaraquemada D. Villanueva J. Scientific Researchers: Antúnez O. Cantero L. Valero L. Scientific Researchers: Díaz R. Odena A. Scientific Researchers: Fernandez M. Mendes M. Scientific Researchers: Aloria K. Beaskoetxea J. Omaetxebarria M J. Scientific Researchers: Navajas R. Paradela A. Scientific Researchers: De la Torre C. Gómez A. Scientific Researchers: De la Torre C. Gómez A. SpHPP Meeting, La Cristalera, Nov 2013 RU3i J. Muñoz CNIO Scientifi c Researc hers: XXXXXX

6. Hemoglobins WD-repeat domains Sox8 Zinc-finger proteins Prrs proteins ZG16B Claudins TMEM proteins Ribosomal proteins RNA binding proteins Ac CoA synthetase RBBP6 IL4R IL21R CD19 LAT Zinc-finger proteins Leukocyte integrins Bromodomain-containing proteins Chromatin remodelling IRX3,5,6 Metallothioneins CDH8 CDH11 CDH5 CDH16 CMTM proteins Zinc-finger proteins Ribosomal proteins CDH3 CDH1 IL-34 Solute carrier proteins CDH13 Forkhead related transcription factors IL-17C SNAI3 CDH15 GAS8 Overview of chromosome 16 protein families Display of proteins according to chromosome position

7. Main protein families in Chr 16 Transcription factors (Zn finger proteins). Nuclear Transcription regulators and chromatin remodelling proteins (embryogenesis). Nuclear RNA binding proteins. Nuclear Ribosomal proteins. Solute carrier proteins. Membrane Secreted proteases (different types) Cadherins. Membrane Secreted chemokines/Cytokines Integrins (a few). Membrane In general, many uncharacterized and poorly known proteins…

8. Main conditions/diseases/alterations affected by chromosome 16 proteins Embryonic development Differentiation Stemness/ regeneration/ pluripotency Spermatogenesis Epithelium-Mesenchymal Transition (Cancer invasion and metastasis) Rare diseases

9. Chr-16 associated diseases (many rare diseases) adenine phosphoribosyltransferase deficiency alpha thalassemia alveolar capillary dysplasia with misalignment of pulmonary veins amyotrophic lateral sclerosis Blau syndrome breast cancer Brody myopathy Brooke-Spiegler syndrome Charcot-Marie-Tooth disease chronic granulomatous disease combined malonic and methylmalonic aciduria complete LCAT deficiency congenital disorder of glycosylation type Ia Crohn disease Ewing sarcoma familial cylindromatosis familial Mediterranean fever Fanconi anemia fatty acid hydroxylase-associated neurodegeneration fish-eye disease Floating-Harbor syndrome giant axonal neuropathy Gitelman syndrome glycogen storage disease type IX Huntington disease-like syndrome infantile neuronal ceroid lipofuscinosis juvenile Batten disease Liddle syndrome lymphangioleiomyomatosis lymphedema-distichiasis syndrome Mainzer-Saldino syndrome malonyl-CoA decarboxylase deficiency Miller syndrome mucolipidosis III gamma mucopolysaccharidosis type IV multiple familial trichoepithelioma North American Indian childhood cirrhosis oculocutaneous albinism osteopetrosis polycystic kidney disease polymicrogyria pseudohypoaldosteronism type 1 pseudoxanthoma elasticum Rubinstein-Taybi syndrome spastic paraplegia type 7 surfactant dysfunction TK2-related mitochondrial DNA depletion syndrome, myopathic form Townes-Brocks Syndrome tuberous sclerosis complex tyrosinemia uromodulin-associated kidney disease http://ghr.nlm.nih.gov/chromosome/16/show/Conditions

10. MCF7 (Breast Epithelial) MCF7 (Breast Epithelial) RAMOS (Lymphoid) RAMOS (Lymphoid) CCD18 (Colon Fibroblast) CCD18 (Colon Fibroblast) SDS-PAGE Basic rpHPLC SDS-PAGE Jurkat (Lymphoid) Jurkat (Lymphoid) SDS-PAGE Basic rpHPLC SDS-PAGE Complete cell extracts were analyzed from: Secretome from KM12 colon cancer cells Around 400 identified proteins in Chr 16 (less than 50%) Only increases 5-6 new proteins the total count

11. New cell sources/ Fractions Embryonic cells (i.e. HEK293) Stem cells, iPSC (mesenchymal, myeloid) (Stem Cell Omics Repository. J. Coon) Testis cells Platelets/ megakaryocytic cell line Brain/ Neural cells Nuclear fractions Membrane Supernatants/ Medium Alternative proteases? Sequence analysis of rare proteins Complete cell extracts from new cell types Design labs for sample preparation and establish common protocols for cell fractionation Cell fractionation will impose some restrictions in the distribution of the materials. Distribute fractions only among a few labs.

12. PTMs analysis Phosphorylation Acetylation Methylation Ubiquitination Glycosilation … There are 15 groups in this area and 5 PTMs, we could try to distribute the groups for PTMs in such a way that each group focuses on 1-2 PTMs to avoid excessive overlap (i.e 4 groups max for each PTM, with the exception of phosphorylation that could be taken by more groups) Implement and standardize protocolos for enrichment and fractionation of each PTM

13. Other activities Quantitative analysis (label-free) between cell lines/tissues Web page for Chr 16 (discovery status, protein atlas, cell location, cell expression …) Tool for batch Chr16 protein assignments Bioinformatics tools for: – Incorporation of isoform (SNPs, Alternative splicing…) sequences and RNAseq to databases – Incorporation of individual proteins/peptides from previous experiments Prepare new manuscripts for HPP

14. Other topics for discussion Criteria for identification Add mitochondria to aim 2? ATMs and SNPs as indicated by proteogenomic studies?

16. Chromosome 16: Differentiation and development chromosome???

18. Fasano A. Physiol Rev 2011;91:151-175