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.• 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.
GroupsCNBCBMCIBPCM-UCMEHU/UPVCIC BIOGUNEIBB-CSICPCB
VHIOCRGCIC-USALUVIDIBELL-IASCNIOCIMA-NAVBIO
• Definition of additional cell lines/tissues• Protocols for subproteome and proteoforms analysis.• Coordinators of the two initiatives and groups involved.
WG 3.-Protein Sequencing Team
RU3aJM. Mato / F. ElortzaCIC-BioGUNE, ProteoRed-ISCIII, Bilbao
Chair: Ignacio CasalCo-chair: Manuel Sánchez del Pino
RU3bJ. AbianIIBB-CSIC ProteoRed-ISCIII, Barcelona
RU3cM. Sánchez del PinoUV, ProteoRed-ISCIII, Valencia
RU3dE. de OliveiraPCB, ProteoRed-ISCIII, Barcelona
RU3eI. CasalCIB-CSIC, ProteoRed-ISCIII, Madrid
RU3fJM. ArizmendiUPV/EHUProteoRed-ISCIII, Bilbao
RU3g JP. AlbarCNB-CSIC, ProteoRed-ISCIII, Madrid
RU3hM.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.
SpHPP Meeting, La Cristalera, Nov 2013
RU3iJ. Muñoz
CNIO
Scientific Researchers:XXXXXX
Hemoglobins
WD-repeat domains
Sox8
Zinc-finger proteins
Prrs proteins
ZG16B
Claudins
TMEM proteins
Ribosomal proteins
RNA binding proteinsAc CoA synthetase
RBBP6
IL4RIL21R
CD19LAT
Zinc-finger proteins
Leukocyte integrins
Bromodomain-containing proteinsChromatin remodelling
IRX3,5,6
Metallothioneins
CDH8CDH11CDH5CDH16
CMTM proteins
Zinc-finger proteins
Ribosomal proteins
CDH3CDH1
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
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…
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
Chr-16 associated diseases (many rare diseases)
adenine phosphoribosyltransferase deficiencyalpha thalassemiaalveolar capillary dysplasia with misalignment of pulmonary veinsamyotrophic lateral sclerosisBlau syndromebreast cancerBrody myopathyBrooke-Spiegler syndromeCharcot-Marie-Tooth diseasechronic granulomatous diseasecombined malonic and methylmalonic aciduriacomplete LCAT deficiencycongenital disorder of glycosylation type IaCrohn diseaseEwing sarcomafamilial cylindromatosisfamilial Mediterranean feverFanconi anemiafatty acid hydroxylase-associated neurodegenerationfish-eye diseaseFloating-Harbor syndromegiant axonal neuropathyGitelman syndrome
glycogen storage disease type IXHuntington disease-like syndromeinfantile neuronal ceroid lipofuscinosisjuvenile Batten diseaseLiddle syndromelymphangioleiomyomatosislymphedema-distichiasis syndromeMainzer-Saldino syndromemalonyl-CoA decarboxylase deficiencyMiller syndromemucolipidosis III gammamucopolysaccharidosis type IVmultiple familial trichoepitheliomaNorth American Indian childhood cirrhosisoculocutaneous albinismosteopetrosispolycystic kidney diseasepolymicrogyriapseudohypoaldosteronism type 1pseudoxanthoma elasticumRubinstein-Taybi syndromespastic paraplegia type 7surfactant dysfunctionTK2-related mitochondrial DNA depletion syndrome, myopathic formTownes-Brocks Syndrometuberous sclerosis complextyrosinemiauromodulin-associated kidney disease
http://ghr.nlm.nih.gov/chromosome/16/show/Conditions
MCF7(Breast
Epithelial)
RAMOS(Lymphoid)
CCD18(Colon
Fibroblast)
• SDS-PAGE
• Basic rpHPLC
• SDS-PAGE
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
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.
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
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
Other topics for discussion• Criteria for identification• Add mitochondria to aim 2?• ATMs and SNPs as indicated by proteogenomic
studies?
Chromosome 16: Differentiation and development chromosome???
Fasano A. Physiol Rev 2011;91:151-175