Genomics on Obesity
Toulouse
7-8 June 2007
PROTEOMICS OF OBESITY
Jennifer RIEUSSET
(jennifer .rieusset@univ-lyon1.fr)
UMR INSERM 870 / INRA 1235
Régulations métaboliques, nutrition et diabètes
Hubert VIDAL
Lyon
CONTENTS
Slide
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 2007
OVERVIEW
What is proteomics ?
Why do it ?
How is it done ?
Application of proteomics to obesity
Genomics on Obesity, Toulouse, 7-8 June 2007
DEFINITIONS
PROTEOME
« The analysis of the entire PROTEin complement expressed
by a genOME, or by a cell or tissue type. »
Wasinger VC et al. Electrophoresis 16 (1995)
PROTEOMICS
Study of the proteins expressed by a genome in a biological sample
(organism, organ, biological fluids), at a given point in time,
in a given situation.
Genomics on Obesity, Toulouse, 7-8 June 2007
Dynamics and protein concentration range
DNA
mRNA
Genome
Transcriptome
Transcription
Proteins
Functional
Proteins
Proteome
Translation
Post-translational
modifications
~300 000 transcripts
~ 3 000 000 proteins
Human:
~ 30 000 genes
Genomics on Obesity, Toulouse, 7-8 June 2007
Diverse properties of proteins
Proteomics is a particularly rich source of biological information
complex
dynamic
PTMs
Genomics on Obesity, Toulouse, 7-8 June 2007
Complexity of proteomes
Same genome
Different proteomes
Genomics on Obesity, Toulouse, 7-8 June 2007
Applications of proteomics
Systematic proteome description
Functional proteomics
Differential analysis (biological markers)
Control
modified
Cell map proteomics (organelles)
protein/protein or protein/drug interactions
Multiprotein complexes
affinity purification
Post-translational modifications
Genomics on Obesity, Toulouse, 7-8 June 2007
OVERVIEW
What is proteomics ?
Why do it ?
How is it done ?
Application of proteomics to obesity
Genomics on Obesity, Toulouse, 7-8 June 2007
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 2007
OVERVIEW
What is proteomic ?
Why do it ?
How is it done ?
Application of proteomics to obesity
Genomics on Obesity, Toulouse, 7-8 June 2007
Proteomics workflow
Sample preparation
Protein separation
Protein detection
Protein identification
Validation and functional analysis
Genomics on Obesity, Toulouse, 7-8 June 2007
Sample preparation
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.
Sample preparation
Protein separation
chaotrope Agents
Urea (5-8M)
Thiourea (2M)
Protein detection
Protein identification
Validation and functional
analysis
Non ionic Detergents
CHAPS (2-4%)
SB 3-10 (2%)
ASB-14 (1%)
Reducing Agents
DTT (65 mM) Dithiothreitol
DTE ( 65 mM) Dithioerythreitol
TBP (2mM) Tributyl phosphine
Ampholytes
IPG buffer pH 3-10
0,5-2%
SDS <0,25%
Genomics on Obesity, Toulouse, 7-8 June 2007
Sample preparation
Lysis procedure
Protease inhibitors
Sample preparation
Protein separation
Protein detection
Removal of interfering substances
 Nucleic acids
 lipids
 salts
Protein identification
 insoluble materials…
Validation and functional
Precipitation
analysis
Fractionation
 subcellular
 differential
Genomics on Obesity, Toulouse, 7-8 June 2007
Protein separation
Sample preparation
Protein separation
Protein detection
Gel-based proteomics
 1D or 2D electrophoresis …
Mass spectrometry driven proteomics
 Chromatography
Protein identification
Validation and functional
analysis
 ICAT …
Protein arrays
Genomics on Obesity, Toulouse, 7-8 June 2007
2D electrophoresis
The most widely used technical approach
1D: separation based on the pI of proteins
2D: separation based on the molecular weight
of proteins
Several visualization/detection possibilities
=> Up to 10 000 protein spots/gel
Genomics on Obesity, Toulouse, 7-8 June 2007
First dimension: IPG strip
IPG: Immobolized pH gradients
Copolymerisation of the pH gradient with the acrylamide matrix on a plastic film
Size :
• Width : 3mm
• Depth: 5mm
• Length: 7, 11, 13, 18 et 24 cm
Best resolution and reproducibility
pH scale:
• large : 7 pH units (3-10, 3-10NL)
• 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)
Large scale
Narrow scale:
Increase
loading capacities
and resolution of
proteins
Genomics on Obesity, Toulouse, 7-8 June 2007
First dimension : Isoelectric focusing (IEF)
IPGphor (Amersham)
Programming :
• Voltage (0-10000V, step-n-hold, gradient)
• 50 µA/strip
• Vh
• temperature: 15-20°C
Genomics on Obesity, Toulouse, 7-8 June 2007
Equilibration
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 2007
SDS-PAGE
3,9
3,9
3,7
3,7
5
5
,,
3
3
7,1
7,1
7,1
8,4
8,4
8,4
pH 3
Poids
moléculair
es
pH
10
8,
8
3,9
7,1
3,7
Criterion cell and precast gels
(BioRad)
8,
8,
88
8,4
5
,
3
Genomics on Obesity, Toulouse, 7-8 June 2007
Protein detection
Sample preparation
Protein separation
Protein detection
Protein identification
Methods
Sensibility
Linearity
Comassie blue
100 ng
low
Silver Nitrate
200 pg
low
Fluorescence
1 ng
high
Fluorescent labelling
250 pg
high
Radiolabelling
1pg
high
Validation and functional
analysis
Genomics on Obesity, Toulouse, 7-8 June 2007
Protein detection
Imagescanner
(Amersham)
Sample preparation
Protein separation
Protein detection
Image Master 2D Platinum
(Amersham)
Protein identification
Validation and functional
analysis
Proteins are automatically detected, background is corrected, spot
density is quantified and spots are matched between up to 100 gels
Genomics on Obesity, Toulouse, 7-8 June 2007
Protein identification
Sample preparation
Protein separation
Protein detection
Protein identification
Validation and functional
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
analysis
Genomics on Obesity, Toulouse, 7-8 June 2007
Validation
Validation
Sample preparation
Protein separation
Protein detection
Protein identification
Validation and functional
analysis
• by Western-blot
• by ELISA
• by activity measurements …
Functional analysis
• Overexpression
• siRNA …
Genomics on Obesity, Toulouse, 7-8 June 2007
OVERVIEW
What is proteomics ?
Why do it ?
How is it done ?
Application of proteomics to obesity
Genomics on Obesity, Toulouse, 7-8 June 2007
OBESITY
Glucose homeostasis
requires the coordinated
actions of various organs
Genomics on Obesity, Toulouse, 7-8 June 2007
Proteomics 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.
Genomics on Obesity, Toulouse, 7-8 June 2007
Proteomics of obesity
Hittel DS et al. Proteome analysis of skeletal muscle from obese and morbidly obese
women. Diabetes. 54:1283-8, 2005.
Genomics on Obesity, Toulouse, 7-8 June 2007
OBESITY
Chronic elevation of NEFAs
Insulin
inhibits insulin action in
skeletal muscle
Genomics on Obesity, Toulouse, 7-8 June 2007
OBESITY
Genomics on Obesity, Toulouse, 7-8 June 2007
Mitochondrial dysfunction
Potential mechanism by which mitochondrial dysfunction induces insulin resistance in skeletal muscle
Lowell BB et al. (2005) Science 307, 384-387.
Genomics on Obesity, Toulouse, 7-8 June 2007
Skeletal muscle
- 4% of muscle mass
- Variation with function of muscles, type of fibers,
physical activity and age.
- 2 populations of mitochondria:
Subsarcolemmal mitochondria
Intermyofibrillar mitochondria
Genomics on Obesity, Toulouse, 7-8 June 2007
Mitochondrial proteome
Human genome: 30 000-40 000 genes
Mitochondria ~ 1500 proteins
Mitochondrial dysfunction
Stockage
Fatty acids
Fatty acids
TG
-
Acyl-CoA
Glucose
Glucose
ß-oxydation
Altered mitochondrial
PGC-1a
structure, biogenesis
and function in skeletal
Target gene
O2
CO2
nucleus
muscle of HFD mice
2D gels of mitochondrial
proteins
Identify 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 2007
Sample preparation
pI
6 SD mice
2 times of diet
(4 and 16 weeks)
3
10
6 HFD mice
Gastrocnemius muscle
pI
3
10
Purification of mitochondria
Protein solubilization
7M Urea, 2M thiourea, 1% ASB14,
2mM TBP, 0.2% IPG buffer, BB
Genomics on Obesity, Toulouse, 7-8 June 2007
2D electrophoresis
6 mt samples SD
6 mt samples HFD
kDa
205
pI
pI
3
10
3
10
80
45
30
1 mt sample SD
1 mt sample HFD
21
14
6.5
SD
6 strip SD
6 strip HFD
IEF
Strip pH 3-10NL
20 µg mt proteins
Active rehydration (50V)
Focalisation: 22 250 V.h.
kDa
pI
3
10
205
80
45
30
21
14
6.5
pI
3
6 2D gels SD
6 2D gels HFD
SDS-PAGE gels: 8-16%
Silver nitrate staining
HFD
Genomics on Obesity, Toulouse, 7-8 June 2007
Data analysis
Format
Image acquisition
(300 dpi)
Resolution (dpi)
depth (8-16 bit)
10
Artefacts
Image analysis
ImageMaster 2D Platinum
Identification by LC-MS/MS
Proteomic platform of Rhônes-Alpes Region
Jerome Garin,
CEA Grenoble
Genomics on Obesity, Toulouse, 7-8 June 2007
Data analysis
Visualizing and calibrating gels
Image acquisition
(300 dpi)
Detecting spots
(intensity, volume)
Matching spots
10
Verification of match
Image analysis
ImageMaster 2D Platinum
Identification by LC-MS/MS
Proteomic platform of Rhônes-Alpes Region
Jerome Garin,
CEA Grenoble
Intra-class analysis
Statistical tests
Kolmogorov, Wilcoxon, T-test
Genomics on Obesity, Toulouse, 7-8 June 2007
Data analysis
Molecules
Ionisation:
MALDI, ESI, …
Image acquisition
(300 dpi)
m/z
Analyser
TOF, Q, B, IT …
10
Identification by LC-MS/MS
Proteomic platform of Rhônes-Alpes Region
Jerome Garin,
CEA Grenoble
Relative intensity (u. a.)
Detection
Image analysis
ImageMaster 2D Platinum
Spectrum
Genomics on Obesity, Toulouse, 7-8 June 2007
Analyse of mRNA and protein expression levels
mRNA=Prot
(46%)
mRNA≠Prot
(54%)
PTMs ?
mRNA: Real-time RT-PCR
Protein: 2D electrophoresis
PROTEOMIC APPROACH
2D Electrophoresis
ProteomLab PF 2D
1st dimension
Isoelectric focalisation
1st dimension
HPLC
Separation based on pI
Separation based on pI
2nd dimension
HPLC
Separation based on hydrophobicity
2nd dimension
SDS-PAGE gel
Separation based on molecular weight
Identification of proteins by mass spectrometry
(Proteomic plateform of Rhône-Alpes Region - JéromeGarin, CEA, Grenoble)
2D electrophoresis
330 matched proteins on 12
gels
C
B
A
D
E
F
18 dysregulated proteins (17 up, 1 down)
A
C
D
E
F
STZ+INS
STZ+INS
STZ
STZ
B
4348
4182
4168
4123
4062
4133 4129
4271
5307 4378
4324 4333 4329
4370 4367
4390
4432
4405
PROTEOMLAB PF2D
Genomics on Obesity, Toulouse, 7-8 June 2007
PF2D: 32KARA
B
Gradient (pH8-4)
A
Genomics on Obesity, Toulouse, 7-8 June 2007
PF2D: ProteoVue
Hydrophobicity profile of
the proteins with
pI 4.96-5.2
Temps de rétention (min)
pH
Washing
A
A 27 26 25 24 23 22
pH Gradient
B
21 20 19 18 17 16 15 14 13 12 11 10
9
B
Wells
Fractions
Genomics on Obesity, Toulouse, 7-8 June 2007
PF2D: DeltaVue
Hydrophobicity profile
for the proteins with
pI 6. 32-6.6
Hydrophobicity profile
for the proteins with
pI 6. 32-6.6
pI
STZ
STZ + INS
STZ + INS
Differential
STZ
Genomics on Obesity, Toulouse, 7-8 June 2007
Sample preparation
1 mg mt proteins
Mitochondria purification
from gastrocnemius muscle
STZ ± INS
49 mitochondrial proteins
are regulated by insulin treatment :
43 and
6
DO 214nm
6 mice/group
n=2
pI 8.05-8.18
pI 8.08-8.21
STZ
STZ + INS
Identification by mass spectrometry
Genomics on Obesity, Toulouse, 7-8 June 2007
Strategy for identification of proteins
PF2D (no MS/MS)
Excision
of bands
MALDI-TOF
Elution
Purification
Differential
analysis
1D gel and
silver nitrate
staining
Genomics on Obesity, Toulouse, 7-8 June 2007
Limits of each proteomic approach
2D-E
2D-LC
time consuming
higher number of proteins
reproductibility
quantity of sample (1-5 mg)
staining
columns/buffers
proteins of high MW
differential analysis
hydrophobic proteins
mass spectrometry
low quantity proteins
several proteins in a fraction
Genomics on Obesity, Toulouse, 7-8 June 2007
Acknowledgements
UMR INSERM U449/INRA U1235
Jennifer Rieusset
Charlotte Bonnard
Hubert Vidal
IFR 62 Laennec
Simone Peyrol
Annabelle Bouchardon
CEA Grenoble
Jérome Garin
CRNH RA
Martine Laville
Genomics on Obesity, Toulouse, 7-8 June 2007
Abbreviations
used
(not otherwise
explained in slides or
notes)
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