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