Lab-On-Chip based protein profiling for CANcer...

Lab-On-Chip based protein profiling for CANcer

DIAgnosisNanoBIO 2008

Barcelona, 10th of June of 2008

Funded by EC contract FP6-034202

NanoBioEuroep 2008. Barcelona 10-06-2008

Manuel Marcelino Perez Perez

Cancer diagnosis


3NanoBioEurope2008 LOCCANDIA

• Histological examination

• Blood test/single biomarker

• Imaging analysis X-rays, CT scans…

Pancreatic cancer



• Detected by expensive diagnostic imaging methods.

• The pancreas

• Poor prognosis at the time of diagnosis

Project goals



•To develop a lab-on-chip for separation and digestion of proteins.

•To develop a full analysis chain integrating bio-nano and info aspects for early detection of pancreatic cancer.

•To test the full analytical chain for early detection of asymptomatic pancreatic cancer patients.

Technical objectives

• To be able to determine low concentration cancer markers in the window of classic cancer marker– Targeted concentration range: 1 to 1000 pmole/L or

1 to 1000 ng/mL• To quantify a multiparametric set of markers to

improve the measurement specificity• To use chromatography nano-columns to improve

the sensitivity• To use electrospray ionisation for a soft on-line

ionisation• To use a mass spectrometry characterisation:

– to get a specific, sensitive and semi-quantitative recognition

– to distinguish isoforms when the sensitivity is appropriate



LOCCANDIA project

6 NanoBioEurope2008 LOCCANDIA


Blood plasma sample

Blood sample

preparation

Lab-on-Chip & mass

spectrometryInformation technology

Selected protein mixture

Peptides spectrogram

Diagnostic information

BIO

Material flow Information flow

NANO INFO

Patient Doctor

Bio part (I)

–Preparation of proteins to make synthetic mixtures using standards protocol.

–Production of the specific antibodies.

–Design of the affinity columns and the quality control of the proteins using MALDI-MS.


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Blood plasma sample

Blood sample

preparation Lab-on-Chip & mass

spectrometry

Selected protein mixture

BIO

Material flow

Patient

LOCCANDIANanoBioEurope2008

Bio part (II)

NanoBioEurope 2008

– MRP8/14, PAP I & p120 – Proteins purification– Antibodies design and production.

LOCCANDIA 8

Lab-on-chip & mass spectrometry

•The objectives are:– to improve the digestion chip– to improve the chromatography-electrospraymicrosystem technology

Funded by EC contract FP6-034202 9

Protein mixture

Peptides spectrogram

Material flow

Lab-on-Chip & mass

spectrometry

NANO

•Two interconnected modules are designed:– a protein digestion module– a liquid chromatography-electrosprayionisation module

NanoBioEuroep 2008 LOCCANDIA

Lab-on-chip& mass spectrometry

• Toward a point of care :

Full system from blood plasma sample to diagnostic information

• Lab-on-Chip

Miniaturized integrated components to increase sensitivity (nano-LC, nano-ESI) and throughput



Lab-on-chip.



– Design of a new nano-liquid chromatography (LC) module including a new e-spray tip.

– New retention phase for chromatography module

- Improvements of the digestion modules.



Chip mounting for Waters Q-TOF mass spectrometer

Interface with MassSpectrometry MS counter-

electrode 0 V

Information technology

To build LOCCANDIA informationmanagement system (LIMS):• a Proteomic Information Management Syste(PIS) for sample information management

• a Clinical Information System (CIS) for patient information management to allow clinical evaluation

Information and data mediation infrastructure including preprocessing, reconstruction, visualization, protein/peptide identification and data analysis modules


Diagnostic information

Information

technology

Peptides spectrogr

am Information flow

INFO

Doctor

17NanoBioEurope2008 Manuel M. Perez-Perez

LIMS modules

Funded by EC contract FP6-034202Manuel M. Pérez 4Manuel Marcelino Perez

To build an Integrated a Clinico-Proteomic Information System (CPIS) for sample information management:• a Data pre-processing & Profile reconstruction module

•an Information and data mediation infrastructure including preprocessing, reconstruction, visualization, protein/peptide identification and data analysis modules

Time alignment

Quantification using V1 or V2

Normalisation using tagged

protein quantity

Selection of the ROI (Region Of Interest) Interpolation Noise

reduction

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Temps de rétention

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nsité

data1_1data1_2data1_3data2_1data2_2data2_3data3_1data3_2data3_3

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0

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Temps de rétention

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nsité

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0.1

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m/z

Temps de retention

m/z

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Temps de retention

m/z

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Suport Vector Machines


4NanoBioEurope2008 Manuel Marcelino Pérez

Source: www.neural-forecasting.com

Example: Mapping from two-dimensional input space with non-linear class boundaries into a three-dimensional feature space with linear separation by hyperplane. Working in high dimensional feature space solves the problem of expressing complex functions.

SVM GAUSSIAN RADIAL BASIS KERNEL

Gaussian Radial Basis kernel function.

Number of Support Vectors : 20 Training error : 0

LOCCANDIA 7NanoBioEurope 2008


NanoBioEurope 2008 Manuel Marcelino Pérez 7

Verification: We build the product RIGHT

Validation: We build the RIGHT product

Verification & validation


Main research outcomes


NanoBioEurope 2008

• an optimized chromatographic-electrospray lab-on-chipdedicated to protein profiling for cancer diagnosis

• an Integrated Clinico-Proteomics Environmentsupporting the integrated device and the diagnosi

• a proof-of-concept of this innovative lab-on-chip technology and the associated analysis chain for cancer diagnosis

LOCCANDIA 19

STRENGTHS & OPPORTUNITIES

• STRENGTHS– Manipulation of sample volumes in the nanoliter range.– High sensitivity– Gel free analytical chain– Advanced biomedical informatics systems– Miniaturization of protein detection processes– Protein quantification using MS and profile

reconstruction algorithm.

• OPPORTUNITIES– Early diagnosis– The concept can be generalized to other cancers as

well as to biomarker discovery.


2ONanoBio2008 LOCCANDIA

List of participants• Atos Origin sae, Spain – ATOS

• Commissariat à l’Energie Atomique,France – CEA-LETI

• DIGILAB BIOVISION GmbH, Germany – DBVN

• Foundation for Research and Technology, Greece – FORTH

• University of Münster, Germany – WWU

• Swiss Institute of Bioinformatics, Switzerland – SIB

• Geneva Bioinformatics, Switzerland - GeneBIO


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ATOS

FORTH

Uni Muenster, Biovision

CEA-LETI

SIB, Gene Bio

WWU and DBVN

CEA-Leti

SIB, GeneBIO

ATOS ORIGIN

FORTH

NanoBioEurope2008 LOCCANDIA

Research Teams

• ATOS Origin: Manuel M Pérez-Pérez, Blanca Jordán, José F. Esteban and Carmen Reina

• CEA-LETI: Pierre Grangeat, Laurent Gerfault, Françoise Vinet, Christine Peponnet, Florence Ricoul, Régis Guillemaud, Grégory Strubel, Caroline Paulus, Nicolas Sarrut and Emeline Mery

• DBVN: Harald Tammen, Karl Schorn and Michael Jurgen

• FORTH: Dimitris Kafetzopoulus, Manolis Tsiknakis, Sophie Kaforou, Hara Roumpaki, George Potamias, Haris Kondylakis, Manolis Kalaitz, Vangelis Kritsotakis

• WWU: Jürgen Schnekeburger, Verena Schick, Jasna Peter-Katalinic, Laura Bindila, Rainer Ossig

• SIB: Frederique Lisacek

• GeneBIO:Pierre Alain Binz


24NanoBioEurope2008 Manuel Marcelino Perez


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5NanoBio 2008 Manuel Marcelino Perez


Plasma

Blood

Solvent Mobile phase

Solvent Mobile phase

Stationary phase

LC –mobile phase– stationary phase

Nano (I)

– Production of new digestion modules.– Design of a new nano-liquid

chromatography (LC) module including a new e-spray tip.

– New retention phase for chromatography module


15NanoBioEurope2008 Manuel M. Perez-Perez

Lab-On-Chip based protein profiling for CANcer...

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