OBEKON - Proteomika

May 22th 2008 Budapest 2

OBEKON - ProteomikaOBEKON - Proteomika

o Richter Genedon NyRto MTAKémiai Kutatóközpont,

Szerkezeti Kémia Intézeto TargetEx Kft.o BioSystems International Kft


(van den Greef, Current Opinion in Chemical Biology 2004, 8:559–565)

Betegség kialakulása biomarkerek diagnoszikumok

Genetic susceptibility

“Multivariate Index Assays”


ÁÁltalltaláános snos séémama

Globális genomika adathalmaz +

Más biológiai klinikai adat

E-Integráció

Hipotézis medált

-molekuláris tudósok- betegellátás

Hipotézis nélküli-genomtudomány-a holnap orvosa Személyreszabott kezelés


LaboratóriumiLaboratóriumi vizsgálatvizsgálat

o Ma- Éhgyomri vércukor- Terheléses vércukor- Koleszterin, Triglicerid

profil- Terheléses vércukor

- antidiabetikum, testmozgás javasolt

- Vércukor kontroll- Sztatin kezelés

o Holnap- Metabolikus státuszII típusú diabétesz

- genetikai prediszpozíció : + LOD >3, Z<0.001, kritikus csúcs 12q31– xx gén

- Diéta hatékonyság P>0.8- Orális antidiabetikum (xx)

hatékonyság P<0.2, yy P>0.8 P450 polimorfizmus SNP (nt 425 AG/AG)

- Szűrés – családtagok >25 év- Sztatin hatékonyság P>0.6


MammaPrint™ *(Agendia) / gen expression profiling for breast cancer

•assesses clearly the individual risk of breast cancer recurrence • provides valuable information to decide on the right treatment • is cleared by the FDA and has been proven in more than 7,000 tests • no additional invasive procedures

ProteomicsProteomics

- global- global- sensitive- sensitiveproteome analysisproteome analysis

OBEKON OBEKON type II diabetes early diagnostics type II diabetes early diagnostics


General Strategy for General Strategy for Proteome CharacterizationProteome Characterization

Purification

Peptides

Mass Spectrometry

Database Search

1-DE 2-DE Solution

Characterization MALDI-TOF MS(peptide mass mapping)

-(LC)-ESI-MS/MS(SEQUEST)

• Identification• PTM• Quantification


Multidimensional Protein Multidimensional Protein Identification Technology (MudPIT)Identification Technology (MudPIT)

SCX RP

Massspectrometer

Identify Proteinsin Mixture

2D ChromatographicSeparation of Peptides

Protein Mixture DigestedPeptides

1.Sample preparation

2. Digest

3. Purificationof peptides

4.Preparationof column

5. 2DLC/LC


Challenges in blood proteome Challenges in blood proteome diagnostics diagnostics

o Six high abundant proteins constitute about 80% of all serum proteins (e.g., albumin alone is about 51%) making virtually impossible to visualize proteins that hold possible important diagnostic information, but present in blood at lower levels.

o Glycoproteins, a subset of blood proteins, can be specially treated and captured for downstream proteomic analysis.


Protein Biomarker technology strategiesProtein Biomarker technology strategies

Biomarker discovery strategies

advantage disadvantage Easy link to clinical assay

2D gel peptide mass fingerprinting

simple Scalability and sensitivity are low

no

Ciphergen protein chip

simple Reproducibility, sensitivity, TOF-MS

is inadequate

no

LC-MS with label(ICAT, SILAC, iTRAC)

Sensitive, but less then ELISA

Expensive,complex to apply to

many samples, insensitive to PTMs, plasma remains a

challenge

no

Peptidomics, sensitive Global nature of the strategy is lost

no

MLAC simple Global nature of the strategy is lost

no


mAB proteomics mAB proteomics workflowworkflow

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


Antibody strategiesAntibody strategies

Antibody mediated biomarker discovery strategies

advantage disadvantage Easy link to clinical assay

Monospecific antibodies

(Uhlen et. al)

scalable Globality is questionable,

reproducibility of polyclonal antibodies,

insensitive to PTMs

yes

Recombinant antibodies and phage discplay

globality Scalability at the screening level,

affinity

no?

BSI’s mAb mediated biomarker

discovery and validation

Sensitive, global, natural antigen

and PTM-s are seen, disease specific

Not apparent(some antibodies may not work with denatured antigen)

yes


Well chosen clinical

collection

Well chosen clinical

collection

Cntrl

Pos.

Enrichment of low

abundance proteins

Enrichment of low

abundance proteins

Prep. of a mAB panel

against virtually

all proteins

Prep. of a mAB panel

against virtually

all proteins

HTS(ELISA, etc.)

HTS(ELISA, etc.)

10,000mAbs

200Primary

Hits

10 Leads

In < 1 yr !

HT MS of biomarker candidate

s

HT MS of biomarker candidate

s

Validation on

clinical collection

Validation on

clinical collection

Diagnostics development,

Clinical drug trial,Regulatory

approval of Dx Kit

Diagnostics development,

Clinical drug trial,Regulatory

approval of Dx Kit

BSI patent pending

Cntrl

Pos.

Assay prototype

Plasmacollection

“Normalized

antigen prep”

+ LC/MS

Hybridoma technology& Screening

Individual ELISAs,Protein ID, Data

integrationBSI- Patent pending

Dx development and test in clinical trial

Theranostics

BSI- Patent pending

Technology WorkflowTechnology Workflow


Normalization of the plasma Normalization of the plasma proteome proteome (BSI patent pending)(BSI patent pending)

Relationship between aggregate number of peptides identified by MS/MS and measured concentration of 76 proteins by quantitative immunoassays is plotted.

David J States, Gilbert S Omenn, Thomas W Blackwell, Damian Fermin, J immy Eng, David W Speicher & Samir M Hanash Nature Biotechnology 24, 333 - 338 (2006)

10 100 1000

0

10

20

30

40

50

nu

mb

er

pe

ptid

es

ide

ntif

ied

by

MS

/MS

reported concentration of proteins in serum (µg/ml)

Agilent depleted High stringency Medium stringency Low stringency

Relationship between aggregate number of peptides identified by MS/MS and the reportedconcentration of the proteins identified in thenormalized samples

Plasmacollection

“Normalized

antigen prep”

+ LC/MS


Individual ELISAs,Protein ID, Data

integrationBSI- Patent pending

Dx development and test in clinical trialBSI- Patent pending


AZ IMMUNIZÁLÁSHOZ FELHASZNÁLT PREPARÁTUMOK TÖMEGSPEKTROMETRIÁS

ANALÍZISE-”PRIMER BIOMARKEREK”

2.ábra: Venn diagramm; Kontrol donorokból és betegekből származó, egy (A) és két (B) lépésben (Agilent és Normalizáló oszlopkromatográfia) depletált plazmamintákból azonosított fehérjék száma. A mindkét csoportban azonosított fehérjék a diagramm átfedő részében láthatóak. Az egyes mintára specifikusak a diagramm két oldalsó részében jelennek meg.

Kontrol plazma

Obez plazma

20 16 26

Kontrol plazma

Obez plazma

18 14 19

A

B


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)

Avera

ge V

maxN

6 E

xperi

ments Sam

e tra

cer

Pool Sam

e d

ay

(SD

of th

e Vm

axN

fro

m the

6 e

xper

imen

ts)

Scatter Plot

VmaxN Control NCI Y12 Tracer 270807-0.5 0 0.5 1 1.5 2

-0.5

0

0.5

1

1.5

2

Scatter Plot

VmaxN Control NCI Y12 Tracer 270807-0.5 0 0.5 1 1.5 2

-0.5

0

0.5

1

1.5

2

Sig

nal w

ith L

C t

race

r (V

max

, no

rm.)

Signal with control tracer (Vmax, norm.)

Screening of the libraries with Screening of the libraries with biotinylated pooled depleted plasma:biotinylated pooled depleted plasma:

LC tracer – pooled 20 patientsLC tracer – pooled 20 patientsControl tracer – pooled 20 matched controlsControl tracer – pooled 20 matched controls

The pooling in order to avoid the biological variability at the initial screening stage

The threshhold (1.5) defined by the sensitivity of the screening assay: direct capture ELISA

The redundancy of the generated mAb libraries is minimized by the immunization procedure and a redundancy screening of the generated

hybridomas

Plasmacollection Plasma prep


Protein I DI mmunoppt.

Peptide arrays

Anti-mouse I gG

Hybridoma supernatant

HRP-streptavidin

Biotinylated plasma protein tracer

Tracers: total plasma depleted plasma normalized plasma


LC diagnostics candidates LC diagnostics candidates

ROC curve - E13NC926M6

1-Specificity

Se

nsi

tivity

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

LC/Ctrl

ROC curve - E13NC926M6

1-Specificity

Se

nsi

tivity

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

NSCLC/Ctrl




Peptide arrays

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

AC Ctrl SCLC squamousCountMedianMeanStdDevMinMax

10 12 5 51.897 1.555 1.384 1.5131.846 1.497 1.285 1.3550.178 0.260 0.363 0.3201.513 1.059 0.713 0.8162.118 1.884 1.590 1.588

set(set(cancer subtype))

avg(V

max n

orm

ali

zed b

y P

C)

for

E12N

C70

E12N70

p < 0.01

AC Ctrl SCLC squamous

p < 0.005

p < 0.005

mAB2


mAb multimarker panel can mAb multimarker panel can correctly classify squamous cell correctly classify squamous cell

carcinomascarcinomas

PCA 1-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

AdenoSquamousSCLCControlPatient with

contradictory cytology/histology

A panel of 4 hybridomas can classify squamous cell carcinoma




Peptide arrays


72

55

40

33

24

17

170130

100

STEP 1:Multi-immunoaffinity

column

1. 2. 3. 4. 5. 6. 7. 8. STD

Visible band

Abscence or low intensity band

STEP 2: Single mAbs

72554033

24

17

170130100

Ft:

Elution:

Plasmacollection

“Normalized antigen prep”

+ LC/ MS


I ndividual ELI SAs,Protein ID, Data

integration


BSI - Patent pending

72554033

24

17

170130100

Eluate

Protein ID-1Protein ID-1


Sequence search against human proteins in SwissProt with the motif KHL(T/S)SA

Motif identification from phage sequences

Eptiope-ID / phage display

E-MAP: Epitope-mediated antigen prediction Bastas et al. (2007) MCP Papers in Press. Published on September 25, 2007 as Manuscript M700107-MCP200

OBEKON Tagetex Kft (Hungary)

Plasmacollection


+ LC/ MS


I ndividual ELI SAs,Protein ID, Data

integration




Multiplex assaysMultiplex assaysLUMINEX PLATFORM

(fluorescence)

Identical results with BSI mABs and tracers on two multiplexing platforms (comparison of singal intensity a set of mABs with two different tracers)

Collaboration with Telechem Inc (CA) , Randox Ltd (IRL). and BMD SAS (France)

Plasmacollection


+ LC/MS




I ndividual ELISAs,Protein ID, Data

integration


CD26 – dipeptidyl peptidase CD26 – dipeptidyl peptidase IVIV

o Mechanism of action was discovered via an unbiased mAB approach

o New generation type II diabetes drug


Kádas János, Élesné Tóth Katalin, Tajcs Veronika, Pál Angéla

BioSystems International Kft, Debrecen, Magyarország

Mariana Kuras William Hempel, Nadege Tardieu, Anne Jullien, Carole Malderez, Yan Kiefert, Paco Samb, Guttman András,

2BioSystems International SAS, Evry, Franciaország

Documents

OBEKON - Proteomika