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O B E K O N Consortium. Application of proteomics methods in the identification of biomarkers, suitable for studying obesity and obesity related diseases. OBECON Consortium. Identifying clinically relevant biomarkers. Biomarker applications. Drug Development. Disease management. - PowerPoint PPT PresentationTRANSCRIPT
02 October 2009 CECON II. Budapest 1
Application of proteomics methods in the Application of proteomics methods in the identification of biomarkers, suitable for studying identification of biomarkers, suitable for studying
obesity and obesity related diseasesobesity and obesity related diseases
O B E K O NO B E K O NConsortium
OBECON Consortium
2
Identifying clinically relevant biomarkers
Select patients to increase likelihood of clinical trial success
Select the best treatment/drug for each patient
Biomarkers as “early killers” or as approved surrogate markers
Improve patient compliance in the absence of early clinical improvement
Differentiate efficacy or safety of a drug within the same class
Select the best treatment/drug for each patient
Biomarkers as “early killers” or used to exclude certain patient groups from clinical trials
Monitor and avoid potential toxic effects
Patient recruitment for clinical trials
Early disease detection, early treatment
Patient recruitment for clinical trials
Predict likely course of disease
Disease managementDisease management
Stratification markers
Stratification markers
Efficacy biomarkersEfficacy biomarkers
Differentiation markers
Differentiation markers
Toxicity biomarkersToxicity biomarkers
Screening markersScreening markers
Prognostic markersPrognostic markers
Drug DevelopmentDrug DevelopmentBiomarker applicationsBiomarker
applications
02 October 2009 CECON II. Budapest
Multivariate panels versus single biomarkers
New tests are likely to increasingly focus on multivariate panels
Multivariate patterns appear more robust than single analyte tests (less sensitive to individual difference and assay noise)
The rational behind is well known in the clinical field (physicians generally integrate multiple test results to arrive to the diagnosis)
Identification of the proteins/analytes in the panel is desirable but not critical
Regulations are currently being put in place (MammaPrint™, FDA regulations on In Vitro Diagnostic Multivariate Index Assays)
02 October 2009 CECON II. Budapest 3
Genome vs. proteomeHuman Genome = 20 - 30,000 genes
Human Proteome = 300,000 to 1,200,000 protein variants
Genome – static; proteome - dinamic; „… there is only a 0.4 correlation between global mRNA and protein
expression..” Metabolic Profiling, ed.G.G.Harrigan, p72, 2004
PTM: Phosphorylation, Acetylation, Methylation, Hydroxy amino acids,
Acylation, Myristic acid, Palmitic acid, Prenylation, Farnesol, Geranylgeranol, Nitrosylation, Oxidation, Other oxidation: loss of SH, Dityrosine formation, Isoaspartate, Glycation variable, Glycoxidation variable, Lipid peroxide adduction variable
Other variations: Presenve of ligand, oligomeric state, cellular localization,
proteolytic form; Protein function determines phenotype:
“Catalytic” proteins, including enzymes, transporters, chaperons, etc.
“Structural” proteins
02 October 2009 CECON II. Budapest
Proteins are probably the best biomarkers
• Proteins are at the site of action (they do the job, catalytic proteins);
• They make up lots of structural elements of the organisms;
• Respond to environmental and pathological changes with modifications;
• The plasma carries the „message” from every tissue and cells;
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Proteome - definitionIf the genome is a list of the instruments in
an orchestra, the proteome is the orchestra playing a symphony.R.Simpson
Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2003,
02 October 2009 CECON II. Budapest
Challanges in proteomics
1. Protein function and activity ;2. What are the differences between the
examination of proteins and nucleic acids? Speed, order of magnitude, sensitivity, reliability;
3. Maybe minor proteins are themost important but their examination is difficult with present day techniques;
4. There is no protein PCR;
02 October 2009 CECON II. Budapest
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Research strategies for proteome-derived biomarkers
• MS-based techniques• reproducibility, sensitivity, dinamic range
and throughput; transformation to clinical test;
• Afinity reagents based approaches• pAB, msAB, mAB, rec(scFV-Fab), scaffold
binders (ankyrin, armadillo, NA);• Antigen-analyte used for immunization and
screening vs, analyte intended to be recognized (peptides, recombinant proteins or domains, PTM, folding, interactions);
• BSI’s approach:• To create nascent mAB libraries;
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Phase
Discovery:Identifyingcandidates
Qualification/Confirmation
ValidationPrototype assay
development
Technology
Product development
Shotgun mass spectrometry
Targeted MS MRM-LC-MS/MS
Prototype immunoassay
ImmunoassaymAb based/multiplexed
1000s
<100
5-25
<5
Number of analytes
Precision
Semi-quantitativeCV 30%
Quantitative
QuantitativeCV 5%
Quantitative CV 3%
Phase
Discovery:Identifyingcandidates
Qualification/Confirmation
ValidationPrototype assay
development
Technology
Product development
Proteome normalizationShotgun
immunization
Global mAB libraries to disease proteome
HTS ELISA screening
ImmunoassaymAb based/multiplexed
10000s
100
10
10
Number of analytes
Precision
QuantitativeCV < 10%
QuantitativeCV 5%
Quantitative CV 3%
Protein ID MS or peptide epitope
Assay developmentMultiplex
multivariate assay
Current, MS based workflow for translation of protein biomarker
discovery into clinical practice
Proteomics strategy of OBECON
02 October 2009 CECON II. Budapest 10
Plasma collection; Represent. sampling
Depletion
Fraction generatio
n
Shotgun MS
LC-MS/MS
mAB-productio
n
Protein N-glyc
Selection differentia
ls
Result verification
on individual samples
Epitope ID
Protein ID
Representative samples from OBECON plasma collectionNOND; OND; OD
02 October 2009 CECON II. Budapest 11
Human plasma-proteins representational inbalance
12
13
Albumin
IgG
Total Plasma Depleted Plasma Normalized PlasmaMARS column
(Agilent)Polyclonal column
Rabbit anti-human serum
BBalancing protein representation in plasma
Multiple Affinity Removal ColumnHu7Hu14
NormalizationUltra-Normalization
Multiple LectinAffinity ColumnMLAC
14
Balancing protein representation in plasma
1----Total plasma
2----Hu7 depleted
3---- Hu7 depleted, norm.
4--- Hu14 depleted
5--- Hu14 depleted, norm.
Proteomics strategy of OBECON
02 October 2009 CECON II. Budapest 15
Plasma collection; Represent. sampling
Depletion
Fraction generatio
n
Shotgun MS
LC-MS/MS
mAB-productio
n
Protein N-glyc
Selection differentia
ls
Result verification
on individual samples
Epitope ID
Protein ID
•alpha-1b-glycoprotein•alpha-2-hs-glycoprotein•apolipoprotein a-i•apolipoprotein c-iii•apolipoprotein e•ceruloplasmin precursor (ferroxidase)•complement c1r•complement c3•complement factor b•hemopexin•thrombin•transthyretin•vitamin d-binding protein•vitronectin
Normalized plasmaControl OBE
Proteomics profiles of the OBE vs Control NP
•apolipoprotein a-ii•chorein 1d•collagen alpha 1(xvii) chain•dorsal neural-tube nuclear protein•fam91a1 protein•filip1l protein•g protein-coupled receptor 45•homeobox protein cux-2•hypothetical protein fp972•inter-alpha-trypsin inhibitor heavy chain 1•kiaa0357•paternally expressed gene 3 isoform 2•scavenger receptor class a, member 3•transcription intermediary factor 1•uncharacterized protein dkfzp667f0711•unnamed protein product 3•vacuolar protein sorting-associated protein 8 homolog•zonadhesin variant 3
•abhydrolase domain-containing protein fam108b1•alpha-2-antiplasmin•anti-oxldl immunoglobulin•complement factor properdin•gelsolin•giant protein p619•heat repeat containing 2•kiaa1662•kininogen•lim and calponin homology domains-containing protein 1•nsp5•nucleoporin nup160•tchp protein•titin•transmembrane protein 108•tubulin•u3 small nucleolar rna-associated protein 15•unnamed protein product 1•unnamed protein product 4
02 October 2009 CECON II. Budapest
Proteomics strategy of OBECON
02 October 2009 CECON II. Budapest 17
Plasma collection; Represent. sampling
Depletion
Fraction generatio
n
Shotgun MS
OGE-IEF, PGC -LC
mAB-productio
n
MS/MS Prot.,N-glyc
Selection differentia
ls
Result verification
on individual samples
Epitope ID
Protein ID
Fractionation of plasma samples by OFFGEL isoelectrophoresis
02 October 2009 CECON II. Budapest 18
M ND D ND D ND D ND D OGE Fr# 5 5 6 6 7 7 8 8
Analysis of complex oligosaccharides using graphitized carbon LC/MS
Glycosilation – PTM;Changes properties of proteins;
Congenital disordersAffects cell-cell communication;Divers and its effect pattern dependent;
Analysis challenging;
02 October 2009 CECON II. Budapest 19
Proteomics strategy of OBECON
02 October 2009 CECON II. Budapest 20
Plasma collection; Represent. sampling
Depletion
Fraction generatio
n
Shotgun MS
LC-MS/MS
mAB-productio
n
Protein N-glyc
Selection differentia
ls
Result verification
on individual samples
Epitope ID
Protein ID
Hybridoma technology
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Hybridoma IDE12NC1 E12... E12NC1... E12NC... E12NC4... E12NC6... E12NC... E12NC905
0.1
0.5
1
5
10
50
100
500
1 000
2 000
Hig
hm
ed
ium
Screening
02 October 2009 CECON II. Budapest 22
Anti-mouse I gG
Hybridoma supernatant
HRP-streptavidin
Biotinylated plasma protein tracer
0.0 0.5 1.0 1.5 2.0 2.5
0.0
0.5
1.0
1.5
2.0
2.5
Comparison of Average VmaxN values (and corresponding SD) between six experiments done with a single tracer, either on the same day or on different days
Average VmaxN 6 Experiments Same tracer Pool Different days(SD of the VmaxN from the 6 experiments)
Ave
rage
Vm
axN
6 E
xper
imen
ts
Sam
e tr
acer
Poo
l Sa
me
day
(SD
of t
he V
max
N fr
om the
6 e
xper
imen
ts)
Capture screening assay: minimal fold change detection is 1.5 (CV<10%) (CV<10%)
Antibodies from hybridoma supernatants recognizing OD...
02 October 2009 CECON II. Budapest 23
Scatter Plot
Sample IDD19OB100 D19OB1101 D19OB25 D19OB420 D19OB585 D19OB765 D19OB901 D20OB574
0
0.5
1
1.5
2
...OND...
02 October 2009 CECON II. Budapest 24
Scatter Plot
Sample IDD19OB100 D19OB1101 D19OB25 D19OB420 D19OB585 D19OB765 D19OB901 D20OB574
0
0.5
1
1.5
... and NOND plasma components.
02 October 2009 CECON II. Budapest 25
Scatter Plot
Sample IDD19OB100 D19OB1101 D19OB25 D19OB420 D19OB585 D19OB765 D19OB901 D20OB574
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
Antibodies can differentially recognize OD vs NOND...
02 October 2009 CECON II. Budapest 26
Scatter Plot
vmaxOBpool48CtrlsHu70 0.5 1 1.5 2 2.5
0
0.5
1
1.5
2
...or OND vs. NOND plasma components.
02 October 2009 CECON II. Budapest 27
Scatter Plot
vmaxOBpool48CtrlsHu70 0.5 1 1.5 2
0
0.5
1
1.5
Cloning selected hybridomas
02 October 2009 CECON II. Budapest 28
Scatter Plot
Hybridoma.Supernatant.IDD16OB172 D16OB172.2 D16OB172.6 D16OB246.12 D16OB246.4 D16OB246.9 D16OB360.3 D16OB360.7 D18J 879
0
0.2
0.4
0.6
0.8
1
OD specific clones
02 October 2009 CECON II. Budapest 29
diabete/NonDiabete
Sample IDD15OB912-D1 D16OB222-D7 D16OB360-D23 D19OB1065-D5 D19OB128-D2 D19OB217-D4 D19OB742-D3 D19OB798-D5
0
20
40
60
80
100
120
140
OND specific clones
02 October 2009 CECON II. Budapest 30
diabete/NonDiabete
Sample IDD15OB912-D1 D16OB222-D7 D16OB360-D23 D19OB1065-D5 D19OB128-D2 D19OB217-D4 D19OB742-D3 D19OB798-D5
0
5
10
15
20
25
30
35
Antibody characterization
Cognate antigen determinationNecessary, because of the immunization
strategy;Protein ID
Immunorecognition conveyed 1-2D gel mediated isolation followed by MS identification;
Epitope ID
02 October 2009 CECON II. Budapest 31
2008.05.08.Scientific meeting 2009
32
Antigen ID: Direct Protein ID Immune recognition
Simple immunoprecipitation Multi-immunoaffinity Protein ID Platform:
Plasma preparation for chromatography Two-step immunoaffinity chromatography
Isolation: One or two dimensional electrophoresis Image analysis/comparison
Identification Antigen ID from gel slices or from eluted fractions via mass
spectrometry
33
Mixture of 8mAb’s
mAb 1mAb 2
mAb 3mAb 4
mAb 8
Plasmapreparation
Multi-immunoaffinityColumn (MIA)
Wash out of non-specific binding proteins
Elution of specific bound antigens
Antigen ID: Direct Protein ID
1 2 3 mAb 8
Wash out of non-specific binding proteins
Elution of specific bound antigen
Individual column chromatography
Image comparison of 1 or 2 dimensional SDS PAGE of flow through and eluate
In gel digestion, LC-MS analysis.
Summary: proteomics method applicationMS-based biomarker discovery
MS-shotgun expt: selectively appearing proteins in the different patient groups;
Analysis of plasma fractions for protein and glycosilation differences in human and in animal models;
Antibody library-based biomarker discoverySix fusions; > 6000 hybridomas;Screening median and high producers using OND,
OD, and NOND plasmas Cloning hybridomas differentiating among groups;Characterization of purified antibodies (E-ID, P-ID); Testing individual plasmas;
02 October 2009 CECON II. Budapest 34
Acknowledgement
OBEKONOBEKONConsortium
02 October 2009 35CECON II. Budapest
Kurucz I.1, Kádas J.1, Tardieu N.2, Malderez C.2, Urbányi Z.3, Drahos L4., Vékey K.4, Flachner B.5, Cseh S.5, Takács L.1,2.
1BioSystems International (BSI) Ltd., Debrecen; 2BSI SAS, Evry, France; 3Gedeon Richter Ltd., Budapest; 4Hungarian Academy of Sciences, Chemical Research Center, Budapest; 5TargetEx Ltd., Dunakeszi;
NKTH # NKFP-A1-2006-0048