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Technologie Biacore et analyse protéomique

Christophe Quétard, Ingénieur d’Application.IIIème Rencontre des plates-formes protéomiques, région PACAMarseille, CIML, 25-26 SEPT 2008

2 / Rencontre Protéomique PACA, CIML - Marseille / 25-26 SEPT 08

Agenda

• Biacore : comment ça marche ?

• Biacore-MS, les différentes approches

• Biacore-MS pour trouver et identifier de nouveaux partenaires

• Biacore-MS, autres applications

• Biacore-MS, liste de références bibliographiques


The Corner-stones of our Technology

IFC Microfluidic System

SPR Detection System

Sensor Chips


Binding and dissociation


A microfluidic developped for subsequentcombination with MSPrecize delivery of sample through syringe pumps and pneumatic valves

Flow cell volume is less than 60nl

Low sample consumption (analyte and ligand)

Efficient mass transport

Measure with confidence off rates as fast as 0.5 s-1

Precise and repeatable concentration measurements

Possibility to reverse the flow

Recovery of material that has been bound in a state suitable for MS analysis

Our Microfluidic has continuously been improved over the past 17 years


What do Biacore systems measure?

Time

Resp

onse

SpecificitySpecificity

How selective?

ConcentrationConcentration

How much active sample?

AffinityAffinity KineticsKinetics

Functionality: How strong? How fast?

ThermodynamicsThermodynamics

What drives the interaction?


Detect and monitor binding interactions

Identify and structurally characterize analytes

Combination:Biacore & MS


BIA/MS Pathways

nanoESIMS-MS

MALDI-TOF-MS

nanoESI

MALDI-TOF-MS

BIABIARemove Sensor Chip from the instrument. Add MALDI matrix

Elute bound analytesfrom the Sensor Chip

BIABIAMS

MS


MS on Eluted and Recovered Analyte

nanoESIMS-MS

MALDI-TOF-MS

nanoESI

20000

20500

21000

21500

22000

22500

23000

23500

24000

24500

25000

0 80 160 240 320 400 480 560 640 720 800Time s

Res

pons

e

RU

desorption

Recovered sample

MS

Analyte Recovery

Biacore® T100 Biacore® 3000


BIACORE®3000 - Surface Prep Unit – Flow cells

Surface Prep, type 2Large area flow cell FCC2

Immobilization in wizard

Used for recovery with wizard/MDL

In Out

extended capture area in type 2 configuration

flow cell area formed with chip in IFC

Immobilization in wizard

Surface Prep, type 1Separate flow cells (1-4) FCC1


Recovery in BIACORE®3000


BIACORE® T100 - Unmatched performance for protein interaction analysis


Recovery Features in BIACORE® T100

• Analytes recovered in small volume (1.5μl)

• Minimum carry-over from sample to recovered solution

• Deposited in vial that contains 10 μl deposition solution (for example trypsin)

• Recovery process defined in Method builder (template)

• Whole process based in one cycle type ”InjectAndRecover”


Martinez et al., 2003. Nature 421(6918): 75-79

Keywords: HDL / atherosclerosis

apoA-1HDL

High affinityHDL receptor

Hepatocyte

Known interaction

Biacore 3000 as a powerful tool

Purpose: to purify this receptor for a subsequent identification

for membrane protein purification


Martinez et al., 2003. Nature 421(6918): 75-79

1 - Total solubilized porcine liver plasma membrane proteins either2 - Purified by affinity chromatography

3 -Purified by Biacore Microrecovery procedure

Biacore SensorgramsapoA-1-bound sensor chips

Ask for figure [email protected]



Advantages of Ligand Fishing by Biacore

Real-time information gives binding capacity control in all steps of the solubilization and purification process

Complex sample mixtures are analyzed

Minimal sample volume required

Control surface(s) readily established

BIACORE detects even low affinity binding events

High quality affinity micropurification using the recovery procedure

Avoids lengthy sample purification before MS analysis

Greatly accelerates the different steps of the purification


Bousquet et al., 2006. EMBO Journal 25: 3943-54

Purpose: to identify novel sst2 interacting partners

Sst2 protein sequence has been screened and a consensus YXXM motif was identified

YXXM motif is known, when Y residue is phosphorylated to bind the PI3K regulatory p85 subunit

Biacore 3000 used to investigate a direct p85 sst2 interaction



Endogenous p85 specifically interact with the pY-peptide




SPR-MS experiment

• Biacore® 3000

• 250 RU of peptides immobilized on 3 flow cells

• Analyte: p85-transfected CHO cell extract

• Total binding response: 2000 RU

• Microrecovery

• Tryptic digestion

• MALDI-TOF

• Unambiguous identification


Key Benefits of Biacore for Functional proteomics/usual affinity purification methods

• 10 000 fold less bait necessary (ng-µg)

• Less non specific binding

• Real-time monitoring of the binding

• Fully automated recovery procedure

• Reproducibility


Biacore-MS fits a large number of otherpurposes like:

• Biomarker discovery

« On-a-chip identification of proteins via revisited BIA-MS strategy »

Boireau et al., 2008. Biosensors and Bioelectronics

• « Use of tandem Biacore-Mass Spectrometry to identify plateletmembrane targets of novel monoclonal antibodies »

C. Ravanat and V. Würtz – EFS Alsace

Biacore Users Meeting, 20-21st of November 2008, IECB - Bordeaux


MS - Directly on the Sensor Chip

Nedelkov, D. and R. W. Nelson (2000). “Practical considerations in BIA/MS: optimising the biosensor-mass spectrometry interface.” J Mol Recognit 13(3): 140-145.

Nedelkov, D., A. Rasooly, et al. (2000). “Multitoxin biosensor-mass spectrometry analysis: a new approach for rapid, real-time, sensitive analysis of Staphylococcal toxins in food.” Int J Food Microbiol 60(1): 1-13.

Nelson, R. W., D. Nedelkov, et al. (2000). “Biomolecular Interaction Analysis Mass Spectrometry. BIA/MS can detect and characterize proteins in complex biological fluids at the low- to subfemtomole level.” Anal Chem 72(11): 404A-411A.

Nelson, R. W., D. Nedelkov. (2003). “Design and use of multi-affinity surfaces in biomolecularinteraction analysis-mass spectrometry (BIA/MS): a step toward the design of SPR/MS arrays.” J Mol Recognit 16(1): 15-19.

Boireau et al., Revisited BIA-MS combination: Entire “on-a-chip” processing leading to the proteins identification at low femtomole to sub-femtomole levels, Biosensor and Bioelectronics (2007), doi:10.1016/j.bios.2008.06.030


MS on Eluted and Recovered Analyte : selection of papers

Natsume, T. et al. 2000 "Combination of biomolecular interaction analysis and mass spectrometric amino acid sequencing” Anal. Chem. 72: 4193-4198.

Kikuchi, J. et al. 2003 “Identification of novel p53-binding proteins by biomolecularinteraction analysis combined with tandem mass spectrometry” Mol Biotechnol 23:203-212

Lopez, F., Pichereaux C., et al. (2003) «Improved sensitivity of biomolecular interaction analysis mass spectrometry for the identification of interacting molecules”Proteomics 3: 402-412

Zhukov et al., 2004. Journal Biomolecular Techniques 15: 112-119

Catimel et al., 2005. Journal of Proteome Research 4: 1646-1656

Buijs and Franklin 2005. Brief Funct Genomic Proteomic 4 (1): 39

Bousquet et al., 2006. The EMBO Journal 25: 3943-54

Larsericsdotter et al., 2006. Proteomics 6, 2355-2364

Nilsson et al., 2007. Journal of Proteome Research

Marchesini et al., 2008. Anal. Chem. 80, 1159-1168

Merci beaucoup

www.biacore.com

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