02 October 2009CECON II. Budapest1 Application of proteomics methods in the identification of...

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)


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


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;

5

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,


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;


8

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;

9

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


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


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.




Depletion

Fraction generatio

n

Shotgun MS

LC-MS/MS

mAB-productio

n

Protein N-glyc


ls

Result verification


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





Depletion

Fraction generatio

n

Shotgun MS

OGE-IEF, PGC -LC

mAB-productio

n

MS/MS Prot.,N-glyc


ls

Result verification


Epitope ID

Protein ID

Fractionation of plasma samples by OFFGEL isoelectrophoresis


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;





Depletion

Fraction generatio

n

Shotgun MS

LC-MS/MS

mAB-productio

n

Protein N-glyc


ls

Result verification


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


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...


Scatter Plot

Sample IDD19OB100 D19OB1101 D19OB25 D19OB420 D19OB585 D19OB765 D19OB901 D20OB574

0

0.5

1

1.5

2

...OND...


Scatter Plot


0

0.5

1

1.5

... and NOND plasma components.


Scatter Plot


-0.2

0

0.2

0.4

0.6

0.8

1

1.2

Antibodies can differentially recognize OD vs NOND...


Scatter Plot

vmaxOBpool48CtrlsHu70 0.5 1 1.5 2 2.5

0

0.5

1

1.5

2

...or OND vs. NOND plasma components.


Scatter Plot

vmaxOBpool48CtrlsHu70 0.5 1 1.5 2

0

0.5

1

1.5

Cloning selected hybridomas


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


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


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


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

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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;


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

02 October 2009CECON II. Budapest1 Application of proteomics methods in the identification of...

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