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WP 3.2

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

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WP 3.2

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

  2. 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. • Definition of additional cell lines/tissues • Protocols for subproteome and proteoforms analysis. • Coordinators of the two initiatives and groups involved. • Protocol and data analysis. Tools. • New strategies October 2013 Nov 2013 • Incorporation of data from new celllines/tissues. • Isolation of subcellularfractions and enrichment test. • Working plan forPTMs. Cells, conditions, groups... Dec 2013 Jan 2014 • SG analysis of subcellularfractions. • SamplepreparationforPTMsanalysis. • PTMs MS analysis Feb2014 March 2014 April2014 • Data analysis and integration. May 2014 Groups CNB CBM CIB PCM-UCM EHU/UPV CIC BIOGUNE IBB-CSIC PCB VHIO CRG CIC-USAL UV IDIBELL-IAS CNIO CIMA-NAVBIO

  3. WG 3.-Protein Sequencing Team Chair: Ignacio Casal Co-chair: Manuel Sánchez del Pino RU3a JM. Mato / F. Elortza CIC-BioGUNE, ProteoRed-ISCIII, Bilbao 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 RU3i J. Muñoz CNIO ScientificResearchers: Azkargorta M. Escobes I. Iloro I. Rodríguez E. ScientificResearchers: Carrascal M. Gallardo O. Jaraquemada D. Villanueva J. ScientificResearchers: Antúnez O. Cantero L. Valero L. ScientificResearchers: Díaz R. Odena A. ScientificResearchers: Fernandez M. Mendes M. ScientificResearchers: Aloria K. Beaskoetxea J. Omaetxebarria M J. ScientificResearchers: Navajas R. Paradela A. ScientificResearchers: De la Torre C. Gómez A. ScientificResearchers: XXXXXX SpHPP Meeting, La Cristalera, Nov 2013

  4. Overview of chromosome 16 proteinfamilies WD-repeatdomains Bromodomain-containingproteins Chromatinremodelling Hemoglobins Ac CoAsynthetase Forkheadrelated transcriptionfactors Sox8 RNA bindingproteins CDH8 CDH11 CDH5 CDH16 CDH3 CDH1 RBBP6 CDH13 CDH15 Zinc-fingerproteins IL-17C CD19 LAT CMTM proteins IL4R IL21R ZG16B IRX3,5,6 GAS8 TMEM proteins IL-34 SNAI3 Prrsproteins Metallothioneins Zinc-fingerproteins Claudins Zinc-fingerproteins Leukocyteintegrins Ribosomalproteins Ribosomalproteins Solutecarrierproteins Display of proteinsaccordingtochromosome position

  5. Mainproteinfamilies in Chr 16 • Transcriptionfactors (Zn fingerproteins). Nuclear • Transcriptionregulators and chromatinremodellingproteins (embryogenesis). Nuclear • RNA bindingproteins. Nuclear • Ribosomalproteins. • Solutecarrierproteins. Membrane • Secretedproteases (differenttypes) • Cadherins. Membrane • Secreted chemokines/Cytokines • Integrins (a few). Membrane • In general, manyuncharacterized and poorlyknownproteins…

  6. Mainconditions/diseases/alterationsaffectedbychromosome 16 proteins • Embryonicdevelopment • Differentiation • Stemness/ regeneration/ pluripotency • Spermatogenesis • Epithelium-MesenchymalTransition (Cancer invasion and metastasis) • Rarediseases

  7. Chr-16 associateddiseases (manyrarediseases) • glycogenstoragediseasetype IX • Huntington disease-likesyndrome • infantile neuronal ceroidlipofuscinosis • juvenileBattendisease • Liddlesyndrome • lymphangioleiomyomatosis • lymphedema-distichiasissyndrome • Mainzer-Saldinosyndrome • malonyl-CoAdecarboxylasedeficiency • Miller syndrome • mucolipidosis III gamma • mucopolysaccharidosistype IV • multiplefamilialtrichoepithelioma • North American Indianchildhoodcirrhosis • oculocutaneousalbinism • osteopetrosis • polycystickidneydisease • polymicrogyria • pseudohypoaldosteronismtype 1 • pseudoxanthomaelasticum • Rubinstein-Taybisyndrome • spasticparaplegiatype 7 • surfactantdysfunction • TK2-related mitochondrial DNA depletionsyndrome, myopathicform • Townes-BrocksSyndrome • tuberoussclerosiscomplex • tyrosinemia • uromodulin-associatedkidneydisease adeninephosphoribosyltransferasedeficiency alphathalassemia alveolar capillarydysplasiawithmisalignment of pulmonaryveins amyotrophic lateral sclerosis Blausyndrome breastcancer Brodymyopathy Brooke-Spieglersyndrome Charcot-Marie-Toothdisease chronicgranulomatousdisease combinedmalonic and methylmalonicaciduria complete LCAT deficiency congenitaldisorder of glycosylationtypeIa Crohn disease Ewing sarcoma familialcylindromatosis familialMediterraneanfever Fanconi anemia fattyacidhydroxylase-associatedneurodegeneration fish-eyedisease Floating-Harbor syndrome giantaxonalneuropathy Gitelmansyndrome http://ghr.nlm.nih.gov/chromosome/16/show/Conditions

  8. Complete cellextractswereanalyzedfrom: CCD18 (Colon Fibroblast) RAMOS (Lymphoid) MCF7 (Breast Epithelial) • SDS-PAGE • SDS-PAGE • Basic rpHPLC • SDS-PAGE Jurkat (Lymphoid) Around 400 identifiedproteins in Chr 16 (lessthan 50%) • SDS-PAGE • Basic rpHPLC • Secretomefrom KM12 colon cancercells Onlyincreases 5-6 new proteinsthe total count

  9. New cellsources/ Fractions • Embryoniccells (i.e. HEK293) • Stemcells, iPSC (mesenchymal, myeloid) (Stem Cell OmicsRepository. J. Coon) • Testiscells • Platelets/ megakaryocyticcellline • Brain/ Neural cells • Nuclear fractions • Membrane • Supernatants/ Medium • Alternativeproteases? Sequenceanalysis of “rare” proteins • Complete cellextractsfrom new celltypes • Designlabs for samplepreparation and establishcommonprotocols for cellfractionation • Cell fractionationwillimposesomerestrictions in thedistribution of thematerials. Distributefractionsonlyamong a fewlabs.

  10. PTMsanalysis • Phosphorylation • Acetylation • Methylation • Ubiquitination • Glycosilation • … • There are 15 groups in thisarea and 5 PTMs, wecould try todistributethegroups for PTMs in such a waythateachgroupfocuseson 1-2 PTMstoavoidexcessiveoverlap (i.e 4 groupsmax for each PTM, withtheexception of phosphorylationthatcould be takenby more groups) • Implement and standardize protocolos for enrichment and fractionation of each PTM

  11. Otheractivities • Quantitativeanalysis (label-free) betweencelllines/tissues • Web page for Chr 16 (discovery status, protein atlas, celllocation, cellexpression …) • Tool for batch Chr16 proteinassignments • Bioinformaticstools for: • Incorporation of isoform (SNPs, Alternativesplicing…) sequences and RNAseqtodatabases • Incorporation of individual proteins/peptidesfrompreviousexperiments • Prepare new manuscripts for HPP

  12. Othertopics for discussion • Criteria for identification • Addmitochondriatoaim 2? • ATMs and SNPs as indicatedbyproteogenomicstudies?

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

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

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