PROTEOMICS OF OBESITY

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


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


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


complex

dynamic

PTMs


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


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


OVERVIEWOVERVIEW


Why do it ?

How is it done ?



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


OVERVIEWOVERVIEW

What is proteomic ?

Why do it ?

How is it done ?



Proteomics workflowProteomics workflow

Sample preparation

Protein separation

Protein detection

Protein identification

Validation and functional analysis


http://www.geerwade.com/gw/images/site/education/grapes.jpg

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




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.


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


Sample preparation

Protein separation

Protein detection



Protein separationProtein separation


Gel-based proteomics

1D or 2D electrophoresis …

Mass spectrometry driven proteomics

Chromatography

ICAT …

Protein arrays

Sample preparation

Protein separation

Protein detection



2D electrophoresis2D electrophoresis


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


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


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


SDS-PAGESDS-PAGE


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


Sample preparation

Protein separation

Protein detection



Protein detectionProtein detection


Image Master 2D PlatinumImage Master 2D Platinum(Amersham)(Amersham)

ImagescannerImagescanner(Amersham)(Amersham)

Sample preparation

Protein separation

Protein detection



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


Sample preparation

Protein separation

Protein detection



ValidationValidation


Validation

• by Western-blot

• by ELISA

• by activity measurements …

Functional analysis

• Overexpression

• siRNA …

Sample preparation

Protein separation

Protein detection



OVERVIEWOVERVIEW


Why do it ?

How is it done ?



OBESITYOBESITY


Glucose homeostasis

requires the coordinated

actions of various organs


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.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15274121&query_hl=4&itool=pubmed_docsum
















































http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15855311&itool=iconfft&query_hl=10&itool=pubmed_docsum















Proteomics of obesityProteomics of obesity


Hittel DS et al. Proteome analysis of skeletal muscle from obese and morbidly obese women. Diabetes. 54:1283-8, 2005.



OBESITYOBESITY


Chronic elevation of NEFAs

inhibits insulin action in

skeletal muscle

InsulinInsulin

OBESITYOBESITY



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


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)


Mitochondrial dysfunction Mitochondrial dysfunction



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



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)


Format

Resolution (dpi)

depth (8-16 bit)

Artefacts

Proteomic platform of Rhônes-Alpes Region

Jerome Garin, CEA Grenoble



10

Identification by LC-MS/MS



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



10

Identification by LC-MS/MS



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


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)


PROTEOMLAB PF2DPROTEOMLAB PF2D


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

)


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


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




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


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


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

PROTEOMICS OF OBESITY

Documents

Transcript of PROTEOMICS OF OBESITY