5 th spanish human proteome project (sphpp) workshop la cristalera, miraflores de la sierra, madrid,...

5th Spanish Human Proteome Project (SpHPP) Workshop

La Cristalera, Miraflores de la Sierra, Madrid, November 5-6th, 2013

SpHPP Council, La Cristalera, Nov 2013

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. BioinformaticsWG5. 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.

WG 1.-Protein Expression, Peptide Standard & Micro

Array Team

RU1a (Protein Micro Array and Protein Expression Team)

Dr. Manuel FuentesCIC/IBMCC (USAL/CSIC), Salamanca

RU1c(Peptide Standard Team)

Dr. Juan P AlbarCNB-CSIC, ProteoRed, Madrid

Scientific Researchers:M. GonzálezN. DasilvaP. DíezM. Pérez de Andrés

Scientific Researchers:M. Lombardía

RU1b (Protein Expression

Team 2)

Dr. Concha GilUCM-PCM,Madrid

Scientific Researchers:M. L. HernáezF. Clemente

RU1e (Bioinformatics,PTM

and cell lines)

Dr. Félix ElortzaCIC-BioGUNEBilbao

WG1. Composition

RU1d (Peptide

Standard Team)

Dr. Eliandre OliveiraPCBBarcelona

Missing proteins

neXtProt: Organizing Protein Knowledge in the Context of Human Proteome Projects

J. Proteome Res. 2013, 12, 293−298Pascale 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

Our Quest for the “Missing Proteins”

638

3844

13359

1071

1216

Uncertain

Awaiting experimental validation

Validated at protein level AND Pep-tideAtlas info

Validated at protein level AND other proteomic info

Validated at protein level AND no proteomics info

1216

Chromosome 16

neXtProt v2013-10-10ENSEMBL v73

UniProtKB v2013_09HPA v11

2360 genes840 protein coding genes143 missing proteins

120 OMIM hitsObesityNeurodegenerative diseasesCancer

Coverage of 73% gene products inLymphoid cellsEpitelial cellsFibroblasts

Shotgun Proteomic

analysis

Transcriptomicanalysis

626 HPA antibodies for Chr16 proteins, 95 for missing

Protein profile Gene expression profileData integration

Global and cell type specific outcomesCorrelation proteome/transcriptomeProteome coverage and chromosome distributionChr16 proteome coverage

Protein expression vectors for 64 Chr16 missing proteins

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.

chromosome entry countprotein level

(PE1)transcript level

(PE2)homology

(PE3)predicted

(PE4)uncertain

(PE5)awaiting protein

validation (P2+P3+P4)1 2061 1589 385 29 9 49 4232 1238 1020 186 3 9 20 1983 1076 871 174 8 3 20 1854 763 614 109 18 2 20 1295 867 691 155 7 4 10 1666 1106 898 164 8 6 30 1787 944 700 179 7 6 52 1928 701 549 97 10 6 39 1139 821 612 152 7 7 43 16610 762 590 147 3 3 19 15311 1319 925 323 24 7 40 35412 1031 841 164 5 2 19 17113 328 264 46 2 6 10 5414 626 502 96 7 3 18 10615 610 451 107 8 3 41 11816 830 656 136 6 1 31 14317 1165 941 185 7 6 26 19818 277 226 40 3 1 7 4419 1425 1009 364 11 4 37 37920 551 435 101 0 1 14 10221 254 165 57 4 0 28 6122 464 355 82 4 2 21 88X 826 604 174 10 6 32 190Y 46 22 16 0 0 8 16

MT 14 13 0 0 0 1 0unknown 40 12 20 4 1 3 25

Description: Chromosome 16 reportName: nextprot_chromosome_16Release: 2013-09-26

NeXtProt DATA

WG1. Unkown proteins clonning workflow

Bacterial selection

Plasmid isolation

DNA sequencing

WG1. Unkown proteins cloning

Chromosome 16 143 Unkown proteins(260 before)

Cloned in pANT7_cGST

WG128 new proteins

Digest with restriction enzymes

Sequencing

Expression (IVTT) and purification (GST)

Mass spectrometry(MRM)

WG1. Unkown proteins expression

List Price 1200 IVTT kit 230 Agarose beads

Sample price: 40 euros

WG1. Unkown proteins analysis workflow I

Expressed protein digestion Skyline peptide/transition selection

MRM assay

WG1. Unkown proteins analysis workflow III

MRM data checking and MSMS spectra validation

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 experiment24 Proteins with identified peptides by MASCOT in MIDAS experiment.

UniprotAccesionnumber

ProteinSelected peptides

Detectedpeptides

Identifiedpeptides

Q8WV35 LRRC29 Leucine-rich repeat-containing protein 29 9 9 9O60359 CACNG3 Voltage-dependent calcium channel gamma-3 subunit 12 9 8Q8TEW6 DOK4 Docking protein 4 11 9 8Q8IUW3 SPATA2L Spermatogenesis-associated protein 2-like protein 9 9 6Q9HBE5 IL21R Interleukin-21 receptor 12 8 5Q8WTQ4 C16ORF78 Uncharacterized protein C16orf78 10 8 2A8K8V0 ZNF785 Zinc finger protein 785 10 7 7Q6PII5 HAGHL Hydroxyacylglutathione hydrolase-like protein 10 7 6Q96H86 ZNF764 Zinc finger protein 764 12 6 6Q96A59 MARVELD3 MARVEL domain-containing protein 3 6 6 6O95976 IGSF6 Immunoglobulin superfamily member 6 9 6 3Q9NZS9 BFAR Bifunctional apoptosis regulator 17 6 2Q8NEV9 IL27 Interleukin-27 subunit alpha 12 6 1Q6UXU4 GSG1L Germ cell-specific gene 1-like protein 7 5 5Q6PL45 C16ORF79 BRICHOS domain-containing protein 5 7 5 0

Q9NWW0 HCFC1R1 Host cell factor C1 regulator 1 7 4 2Q8N635 C16ORF73 Meiosis-specific with OB domain-containing protein 14 4 1Q96B96 TMEM159 Promethin 3 3 2Q8TDN1 KCNG4 Potassium voltage-gated channel subfamily G member 4 11 3 2Q8IYM1 SEP12 Septin-12 15 3 2Q8IZF4 GPR114 Probable G-protein coupled receptor 114 11 3 1P17023 ZNF19 Zinc finger protein 19 15 3 0O75324 SNN Stannin 3 2 1Q9NXF8 ZDHC7 Palmitoyltransferase ZDHHC7 7 2 2Q8TAZ6 CKLFSF2 CKLF-like MARVEL transmembrane domain-containing protein 2 6 2 1Q8TB05 FAM100A UBA-like domain-containing protein 1 1 1 1Q9BTX3 TMEM208 Transmembrane protein 208 3 1 0Q8WVE7 TMEM170A Transmembrane protein 170A 6 1 0

WG1. Location of unkown proteins

Protein Location ExpressionLRRC29 Leucine-rich repeat-containing protein 29 Nucleus Expressed in heart, kidney, liver, lung and pancreasCACNG3 Voltage-dependent calcium channel gamma-3 subunit MB

DOK4 Docking protein 4 MBWidely expressed. High expression in skeletal muscle, heart, kidney and liver.

SPATA2L Spermatogenesis-associated protein 2-like proteinNucleus, mitochondria

widely expressed

IL21R Interleukin-21 receptor MBSelectively expressed in lymphoid tissues. Most highly expressed in thymus and spleen.

C16ORF78 Uncharacterized protein C16orf78 CitoplasmicZNF785 Zinc finger protein 785 NucleusHAGHL Hydroxyacylglutathione hydrolase-like proteinZNF764 Zinc finger protein 764 NucleusMARVELD3 MARVEL domain-containing protein 3 MB

IGSF6 Immunoglobulin superfamily member 6 MBExpressed in peripheral blood lymphocytes, spleen and lymph node, with low levels in thymus, bone marrow and fetal liver

BFAR Bifunctional apoptosis regulatorEndoplasmic reticulum

Expressed highly in brain, moderately in small intestine, weakly in testes and only faintly in liver and skeletal muscle

IL27 Interleukin-27 subunit alpha SecretedGSG1L Germ cell-specific gene 1-like protein MBC16ORF79 BRICHOS domain-containing protein 5 MB Prostate, pancreas, salivary glandsHCFC1R1 Host cell factor C1 regulator 1 MBC16ORF73 Meiosis-specific with OB domain-containing protein MB

TMEM159 Promethin MBExpressed at high levels in the heart and skeletal muscle. Expressed at low levels in kidney, small intestine, lung and liver.

KCNG4 Potassium voltage-gated channel subfamily G member 4 MBHighly expressed in brain, and at lower levels in liver, small intestine and colon.

SEP12 Septin-12 cytoskeleton Widely expressedGPR114 Probable G-protein coupled receptor 114 MBZNF19 Zinc finger protein 19 Nucleus

SNN Stannin Mitochondrion outer membrane

ZDHC7 Palmitoyltransferase ZDHHC7 GolgiCKLFSF2 CKLF-like MARVEL transmembrane domain-containing protein 2

MB Highly expressed in testis

FAM100A UBA-like domain-containing protein 1

TMEM208 Transmembrane protein 208Endoplasmic reticulum membrane

TMEM170A Transmembrane protein 170A MB

ProteinMissingprotein

LRRC29 Leucine-rich repeat-containing protein 29 YESCACNG3 Voltage-dependent calcium channel gamma-3 subunit YESDOK4 Docking protein 4 No Expression PASPATA2L Spermatogenesis-associated protein 2-like protein No Proteomics and expression Two proteotipic peptides. WB, PAIL21R Interleukin-21 receptor No Expression WB, PAC16ORF78 Uncharacterized protein C16orf78 YESZNF785 Zinc finger protein 785 YESHAGHL Hydroxyacylglutathione hydrolase-like protein No Proteomics One proteotipic peptideZNF764 Zinc finger protein 764 No Proteomics and expression One proteotipic peptide. WB, PAMARVELD3 MARVEL domain-containing protein 3 No Proteomics and expression Three proteotipic peptides. PAIGSF6 Immunoglobulin superfamily member 6 No Proteomics and expression One proteotipic peptide. PABFAR Bifunctional apoptosis regulator No Proteomics and expression Four proteotipic peptides. WBIL27 Interleukin-27 subunit alpha No Expression OthersGSG1L Germ cell-specific gene 1-like protein YESC16ORF79 BRICHOS domain-containing protein 5 YESHCFC1R1 Host cell factor C1 regulator 1 No ExperimentalC16ORF73 Meiosis-specific with OB domain-containing protein YESTMEM159 Promethin No Proteomics and expression Three proteotipic peptides. WBKCNG4 Potassium voltage-gated channel subfamily G member 4 YESSEP12 Septin-12 No Expression IF, WB, PAGPR114 Probable G-protein coupled receptor 114 YESZNF19 Zinc finger protein 19 YESSNN Stannin No 3DZDHC7 Palmitoyltransferase ZDHHC7 No ExperimentalCKLFSF2 CKLF-like MARVEL transmembrane domain-containing protein 2

YES

FAM100A UBA-like domain-containing protein 1 No Proteomics One proteotipic peptideTMEM208 Transmembrane protein 208 No Proteomics and expression One proteotipic peptide. WB, PATMEM170A Transmembrane protein 170A No Proteomics One proteotipic peptide

Evidence

WG1. Unknown proteins

PA: protein array, WB: western blot, IF: immunofluorescency

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.

GroupsCNBUCM-PCMPCBCIC-BIOGUNECIC-USAL

Other groups might also participate in defining the best biological matrix and also in the interlaboratory validation process.

Dec 2014

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

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)

GroupsCNBUCM-PCMPCBCIC-BIOGUNECIC-USAL

Other groups might also participate in defining the best biological matrix and also in the interlaboratory validation process.

WG1. Unkown proteins results. In detail

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)

• 66 cell types• 48 tissues• Clinical specimens

5 th spanish human proteome project (sphpp) workshop la cristalera, miraflores de la sierra, madrid,...

Documents

protein knowledge

protein level

madrid scientific researchers

expression of missing

andrs scientific researchers

selected cell lines

missing proteins nextprot

socalled missing proteins