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Genomics on Obesity Toulouse 7-8 June 2007. PROTEOMICS OF OBESITY. Jennifer RIEUSSET (jennifer [email protected]). UMR INSERM 870 / INRA 1235 Régulations métaboliques, nutrition et diabètes Hubert VIDAL Lyon. CONTENTS. Slide. What is proteomics ? 3 - 8 - PowerPoint PPT Presentation
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PROTEOMICS OF OBESITY
UMR INSERM 870 / INRA 1235Régulations métaboliques, nutrition et diabètes
Hubert VIDALLyon
Jennifer RIEUSSETJennifer RIEUSSET(jennifer [email protected])(jennifer [email protected])
Genomics on ObesityToulouse
7-8 June 2007
CONTENTSCONTENTS
What is proteomics ? 3 - 8
Why do it ? 9 - 10
How is it done ? 11 - 24
Application of proteomics to obesity 25 – 49
Acknowledgements 50
Abbreviations used 51
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Slide
OVERVIEWOVERVIEW
What is proteomics ?
Why do it ?
How is it done ?
Application of proteomics to obesity
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
DEFINITIONSDEFINITIONS
« The analysis of the entire « The analysis of the entire PROTEPROTEin complement expressedin complement expressedby a genby a genOMEOME, or by a cell or tissue type. », or by a cell or tissue type. »
Wasinger VC et al. Electrophoresis 16 (1995)Wasinger VC et al. Electrophoresis 16 (1995)
PROTEOME
Study of the proteins expressed by a genome in Study of the proteins expressed by a genome in a biological samplea biological sample(organism, organ, biological fluids), at (organism, organ, biological fluids), at a given point in timea given point in time, ,
in in a given situationa given situation..
PROTEOMICS
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Dynamics and protein concentration rangeDynamics and protein concentration range
DNA mRNA ProteinsFunctionalProteins
Genome Transcriptome Proteome
Transcription TranslationPost-translational
modifications
Human:
~ 30 000 genes ~300 000 transcripts ~ 3 000 000 proteins
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
complex
dynamic
PTMs
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Diverse properties of proteinsDiverse properties of proteins
Proteomics is a particularly rich source of biological information
Same genomeSame genome
Different proteomesDifferent proteomes
Complexity of proteomesComplexity of proteomes
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Applications of proteomicsApplications of proteomics
Systematic proteome description
Functional proteomics
Differential analysis (biological markers)
Cell map proteomics (organelles)
protein/protein or protein/drug interactions
Post-translational modifications
Control modified
Multiprotein complexes affinity purification
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
OVERVIEWOVERVIEW
What is proteomics ?
Why do it ?
How is it done ?
Application of proteomics to obesity
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Why do proteomics ?Why do proteomics ?
mRNA expression analysis does not always reflect the expression level
of proteins
Biological samples such as CSF, serum, urine etc. are not suitable for
mRNA expression analysis
It focuses on gene products – the active agents in cells/tissues/organisms
Analyse the modifications of proteins that are not apparent from DNA
sequence (i.e. post-translational modifications)
Analyse the location of proteins
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
OVERVIEWOVERVIEW
What is proteomic ?
Why do it ?
How is it done ?
Application of proteomics to obesity
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Proteomics workflowProteomics workflow
Sample preparation
Protein separation
Protein detection
Protein identification
Validation and functional analysis
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Sample preparationSample preparation
chaotrope Agents Reducing Agents
Non ionic DetergentsAmpholytes
Urea (5-8M)Urea (5-8M)Thiourea (2M)Thiourea (2M)
CHAPS (2-4%)CHAPS (2-4%)SB 3-10 (2%)SB 3-10 (2%)ASB-14 (1%) ASB-14 (1%)
SDS <0,25%SDS <0,25%
DTT (65 mM) DithiothreitolDTT (65 mM) DithiothreitolDTE ( 65 mM) DithioerythreitolDTE ( 65 mM) DithioerythreitolTBP (2mM) Tributyl phosphineTBP (2mM) Tributyl phosphine
IPG buffer pH 3-10IPG buffer pH 3-100,5-2%0,5-2%
Sample preparation
Protein separation
Protein detection
Protein identification
Validation and functional analysis
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Conditions sufficiently denaturing to solubilize a maximum of proteins, to dissociate all the complexes,Conditions sufficiently denaturing to solubilize a maximum of proteins, to dissociate all the complexes,to maintain them in solution and avoid all chemical modifications of protein subunits.to maintain them in solution and avoid all chemical modifications of protein subunits.
Sample preparationSample preparation
Lysis procedureLysis procedure
Protease inhibitors Protease inhibitors
Removal of interfering substances Removal of interfering substances
Nucleic acidsNucleic acids
lipidslipids
saltssalts
insoluble materials…insoluble materials…
PrecipitationPrecipitation
FractionationFractionation
subcellularsubcellular
differentialdifferential
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Sample preparation
Protein separation
Protein detection
Protein identification
Validation and functional analysis
Protein separationProtein separation
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Gel-based proteomics
1D or 2D electrophoresis …
Mass spectrometry driven proteomics
Chromatography
ICAT …
Protein arrays
Sample preparation
Protein separation
Protein detection
Protein identification
Validation and functional analysis
2D electrophoresis2D electrophoresis
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
1D: separation based on the pI of proteins1D: separation based on the pI of proteins
2D: separation based on the molecular weight 2D: separation based on the molecular weight of proteinsof proteins
Several visualization/detection possibilitiesSeveral visualization/detection possibilities
The most widely used technical approachThe most widely used technical approach
=> Up to 10 000 protein spots/gel=> Up to 10 000 protein spots/gel
First dimension: IPG stripFirst dimension: IPG strip
Size :Size : • Width : 3mmWidth : 3mm• Depth: 5mmDepth: 5mm• Length: Length: 7, 11, 13, 18 et 24 cm7, 11, 13, 18 et 24 cm
pH scale:pH scale:
• large : 7 pH units (3-10, 3-10NL)large : 7 pH units (3-10, 3-10NL)
• narrow : 3-4 pH units (3-7, 4-7, 6-9, 6-11)narrow : 3-4 pH units (3-7, 4-7, 6-9, 6-11)
• micro : 1 pH unit (3,5-4,5, 4-5, 4,5-5,5, 5-6, 5,5-6,5)micro : 1 pH unit (3,5-4,5, 4-5, 4,5-5,5, 5-6, 5,5-6,5)
Narrow scale:Narrow scale:Increase Increase
loading capacitiesloading capacitiesand resolution of and resolution of
proteinsproteins
Large scaleLarge scale
Best resolution and reproducibility
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
IPG: Immobolized pH gradientsIPG: Immobolized pH gradientsCopolymerisation of the pH gradient with the acrylamide matrix on a plastic filmCopolymerisation of the pH gradient with the acrylamide matrix on a plastic film
First dimension :First dimension : Isoelectric focusing (IEF)Isoelectric focusing (IEF)
IPGphor (Amersham)
Programming :Programming :• Voltage (0-10000V, step-n-hold, gradient)Voltage (0-10000V, step-n-hold, gradient)• 50 µA/strip50 µA/strip• Vh Vh • temperature: 15-20°Ctemperature: 15-20°C
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
EquilibrationEquilibration
Tris-HCl 50 mM pH 8,8, Urée 6M, Glycérol 30%, SDS 2%+ DTT 125 mM during10 min+ iodoacétamide 125 mM during 10 min
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
SDS-PAGESDS-PAGE
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Criterion cell and precast gels(BioRad)
8,88,83,93,9
5,3
5,3
3,73,7 7,17,17,18,48,48,4
pH 3 pH 10
Poids moléculair
es 3,9
3,75,3
7,1 8,4
8,8
Protein detectionProtein detection
MethodsMethods
Comassie blueComassie blue
Silver NitrateSilver Nitrate
FluorescenceFluorescence
Fluorescent labellingFluorescent labelling
RadiolabellingRadiolabelling
SensibilitySensibility
100 ng100 ng
200 pg200 pg
1 ng1 ng
250 pg250 pg
1pg1pg
LinearityLinearity
lowlow
lowlow
highhigh
highhigh
highhigh
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Sample preparation
Protein separation
Protein detection
Protein identification
Validation and functional analysis
Protein detectionProtein detection
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Image Master 2D PlatinumImage Master 2D Platinum(Amersham)(Amersham)
ImagescannerImagescanner(Amersham)(Amersham)
Sample preparation
Protein separation
Protein detection
Protein identification
Validation and functional analysis
Proteins are automatically detected, background is corrected, spotdensity is quantified and spots are matched between up to 100 gels
MS identification of proteins after quantitative analysis
by 2DE
• Peptide mass fingerprinting (MALDI MS)
• Sequence based identification (MS/MS)
Identification and quantitation using MS
• Labelling samples for quantitative analysis
• Identification of post-translational modifications
Protein identificationProtein identification
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Sample preparation
Protein separation
Protein detection
Protein identification
Validation and functional analysis
ValidationValidation
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Validation
• by Western-blot
• by ELISA
• by activity measurements …
Functional analysis
• Overexpression
• siRNA …
Sample preparation
Protein separation
Protein detection
Protein identification
Validation and functional analysis
OVERVIEWOVERVIEW
What is proteomics ?
Why do it ?
How is it done ?
Application of proteomics to obesity
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
OBESITYOBESITY
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Glucose homeostasis
requires the coordinated
actions of various organs
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Proteomics of obesityProteomics of obesity
Schmid GM, Converset V, Walter N, Sennitt MV, Leung KY, Byers H, Ward M, Hochstrasser DF, Cawthorne MA, Sanchez JC. Effect of high-fat diet on the expression of proteins in muscle, adipose tissues, and liver of C57BL/6 mice. Proteomics. 4:2270-82, 2004.
Sanchez JC, Converset V, Nolan A, Schmid G, Wang S, Heller M, Sennitt MV, Hochstrasser DF, Cawthorne MA.Effect of rosiglitazone on the differential expression of obesity and insulin resistance associated proteins in lep/lep mice. Proteomics. 3:1500-20, 2003.
Budde P, Schulte I, Appel A, Neitz S, Kellmann M, Tammen H, Hess R, Rose Peptidomics biomarker discovery in mouse models of obesity and type 2 diabetes. Comb Chem High Throughput Screen. 8:775-81, 2005.
Hittel DS, Hathout Y, Hoffman EP, Houmard JA. Proteome analysis of skeletal muscle from obese and morbidly obese women. Diabetes. 54:1283-8, 2005.
DeLany JP, Floyd ZE, Zvonic S, Smith A, Gravois A, Reiners E, Wu X, Kilroy G, Lefevre M, Gimble JM. Proteomic analysis of primary cultures of human adipose-derived stem cells: modulation by Adipogenesis.Mol Cell Proteomics. 4:731-40, 2005.
Xu A, Wang Y, Xu JY, Stejskal D, Tam S, Zhang J, Wat NM, Wong WK, Lam KS. Adipocyte fatty acid-binding protein is a plasma biomarker closely associated with obesity and metabolic syndrome. Clin Chem. 52:405-13, 2006.
Proteomics of obesityProteomics of obesity
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Hittel DS et al. Proteome analysis of skeletal muscle from obese and morbidly obese women. Diabetes. 54:1283-8, 2005.
OBESITYOBESITY
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Chronic elevation of NEFAs
inhibits insulin action in
skeletal muscle
InsulinInsulin
OBESITYOBESITY
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Mitochondrial dysfunction Mitochondrial dysfunction
Lowell BB et al. (2005) Science 307, 384-387.
Potential mechanism by which mitochondrial dysfunction induces insulin resistance in skeletal musclePotential mechanism by which mitochondrial dysfunction induces insulin resistance in skeletal muscle
Skeletal muscleSkeletal muscle
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Subsarcolemmal mitochondria
Intermyofibrillar mitochondria
- 4% of muscle mass- 4% of muscle mass
- Variation with function of muscles, type of fibers,- Variation with function of muscles, type of fibers,
physical activity and age.physical activity and age.
- 2 populations of mitochondria:- 2 populations of mitochondria:
Human genome: Human genome: 30 000-40 00030 000-40 000 genes genes Mitochondria ~ Mitochondria ~ 15001500 proteins proteins
Mitochondrial proteomeMitochondrial proteome
Stockage
TGFatty acids
Acyl-CoA
O2CO2
ß-oxydation
Signaling
-Fatty acids
PGC-1
nucleus
2D gels of mitochondrial proteins
GlucoseGlucose
Target gene
Altered mitochondrial
structure, biogenesis
and function in skeletal
muscle of HFD miceIdentify differentially expressed
proteins in skeletal muscle of SD and HFD-fed mice
(after 4 and 16 weeks of diet)
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Mitochondrial dysfunction Mitochondrial dysfunction
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Sample preparationSample preparation
6 SD mice
10pI 3
10pI 3
6 HFD mice
2 times of diet (4 and 16 weeks)
Gastrocnemius muscle
Purification of mitochondria
Protein solubilization 7M Urea, 2M thiourea, 1% ASB14, 2mM TBP, 0.2% IPG buffer, BB
pI 3
10pI 3
IEF
Strip pH 3-10NL20 µg mt proteinsActive rehydration (50V)Focalisation: 22 250 V.h.
SDS-PAGE gels: 8-16%Silver nitrate staining
6 mt samples SD6 mt samples HFD
1 mt sample SD1 mt sample HFD
6 strip SD6 strip HFD
6 2D gels SD6 2D gels HFD HFD
10pI 3kDa
205
45
30
21
14
6.5
80
10pI 3kDa
205
45
30
21
14
6.5
80
SD
2D electrophoresis2D electrophoresis
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Data analysisData analysis
Image analysis ImageMaster 2D Platinum
10
Identification by LC-MS/MS Proteomic platform of Rhônes-Alpes Region Jerome Garin, CEA Grenoble
Image acquisition(300 dpi)
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Format
Resolution (dpi)
depth (8-16 bit)
Artefacts
Proteomic platform of Rhônes-Alpes Region
Jerome Garin, CEA Grenoble
Data analysisData analysis
Image analysis ImageMaster 2D Platinum
10
Identification by LC-MS/MS
Image acquisition(300 dpi)
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Visualizing and calibrating gels
Detecting spots (intensity, volume)
Matching spots
Verification of match
Intra-class analysis
Statistical tests Kolmogorov, Wilcoxon, T-test
Proteomic platform of Rhônes-Alpes Region
Jerome Garin, CEA Grenoble
Data analysisData analysis
Image analysis ImageMaster 2D Platinum
10
Identification by LC-MS/MS
Image acquisition(300 dpi)
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Molecules
Ionisation:MALDI, ESI, …
Rel
ativ
e in
tens
ity
(u.
a.)
Analyser
TOF, Q, B, IT …
Spectrum
m/z
Detection
Analyse of mRNA and protein expression levelsAnalyse of mRNA and protein expression levels
mRNA=Prot(46%)
mRNA≠Prot(54%)
mRNA: Real-time RT-PCR
Protein: 2D electrophoresis
PTMs ?
1st dimensionIsoelectric focalisation
Separation based on pI
2nd dimension SDS-PAGE gel
Separation based on molecular weight
1st dimensionHPLC
Separation based on pI
2nd dimensionHPLC
Separation based on hydrophobicity
ProteomLab PF 2DProteomLab PF 2D
Identification of proteins by mass spectrometry
(Proteomic plateform of Rhône-Alpes Region - JéromeGarin, CEA, Grenoble)
2D Electrophoresis2D Electrophoresis
PROTEOMIC APPROACHPROTEOMIC APPROACH
2D electrophoresis2D electrophoresis
A D
B C
FE
ST
ZS
TZ
+IN
S
A DB C
4348 4062 4123 4133 4129
4182 4168 4271 5307 4378
FE
ST
ZS
TZ
+IN
S
44054324 4333 4329 4370 4367
4390 4432
330 matched proteins on 12 gels
18 dysregulated proteins (17 up, 1 down)
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
PROTEOMLAB PF2DPROTEOMLAB PF2D
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
PF2D: 32KARAPF2D: 32KARA
B AGradient (pH8-4)
Washing A pH Gradient B
Hyd
rop
hob
icit
y
pH
WellsFractionsB91011121314151617181920212223242526A 27
Tem
ps
de
réte
nti
on (
min
)
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
PF2D: ProteoVuePF2D: ProteoVue
Hydrophobicity profile of the proteins with
pI 4.96-5.2
Hyd
roph
obic
ité
pI
STZSTZ + INS Differential
STZSTZ + INS
Hydrophobicity profile for the proteins with
pI 6. 32-6.6
Hydrophobicity profile for the proteins with
pI 6. 32-6.6
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
PF2D: DeltaVuePF2D: DeltaVue
STZ ± INS
Mitochondria purificationfrom gastrocnemius muscle
1 mg mt proteins
STZ
STZ + INS
DO
214
nm
pI 8.05-8.18pI 8.08-8.21
6 mice/groupn=2
49 mitochondrial proteins49 mitochondrial proteins
are regulated by insulin treatment :are regulated by insulin treatment :
43 and 643 and 6
Identification by mass spectrometry
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Sample preparationSample preparation
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Strategy for identification of proteinsStrategy for identification of proteins
PurificationPF2D (no MS/MS) Elution
1D gel and silver nitrate staining
Differential analysis
Excision of bands
MALDI-TOF
2D-E 2D-LC
time consuming
reproductibility
staining
proteins of high MW
hydrophobic proteins
low quantity proteins
higher number of proteins
quantity of sample (1-5 mg)
columns/buffers
differential analysis
mass spectrometry
several proteins in a fraction
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
Limits of each proteomic approachLimits of each proteomic approach
UMR INSERM U449/INRA U1235Jennifer RieussetCharlotte Bonnard
Hubert Vidal
IFR 62 LaennecSimone Peyrol
Annabelle Bouchardon
CEA GrenobleJérome Garin
CRNH RAMartine Laville
Genomics on Obesity, Toulouse, 7-8 June 2007Genomics on Obesity, Toulouse, 7-8 June 2007
AcknowledgementsAcknowledgements
Abbreviations Abbreviations usedused
((not otherwise not otherwise explained in slides or explained in slides or
notesnotes))
ADN = DNA
ARN = RNA
ASB Aminosulphobetaine (detergent)
CHAPS 3-[(3-cholamido propyl) dimethyl ammonio]-1-propanesulphonate (detergent)
COX Cytochrome C oxidase
CSF Cerebrospinal fluid
ELISA Enzyme-linked immunosorbent assay
GTT Glucose tolerance test
HFD High fat diet
HPRT Hypoxanthine/guanine phosphoribosyl transferase (a housekeeping gene)
ICAT Isotope-coded affinity tagging
IPG (buffer) Immobilized pH gradient
LC Liquid chromatography
MALDI-TOF Matrix assisted laser desorption/ionization – time of flight (mass spectrometry)
MS Mass spectrometry
mt Mitochondria(l)
PGC (-1α, etc) Peroxisome-proliferator-activated receptor-gamma co-activator
PTM Post-translational modification (of proteins)
SB Sulphobetaine (detergent)
SD Standard diet
SDS-PAGE Sodium dodecyl sulfate – polyacrylamide gel electrophoresis
siRNA Small interfering RNA
STZ Streptozotocin