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Francisco J. Blanco, M.D.Rheumatology Division

Proteomic Lab-Proteo-RedHospital Universitario A Coruña

A Coruña-SPAIN

PROTEOMICS APPROACH FOR THE SEARCH OF OA BIOMARKERS IN SERUM

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OA: Challenges• Pathogenesis

• Diagnostic: Biomarkers

• Treatment

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Treatment: OA Models• Treatment of Human OA• Treatment of OA Animal model

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Diagnosis of Early OA vs Later OA

SILENT DISEASE SYMPTOMATIC DISEASE

JOINTMETABOLIC

ABNORMALITY

Pre-RADRIOGRAPHIC PAINFUL RADIOGRAPHIC OA

US

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Proteomics of Human Joint

Ruiz-Romero and FJ Blanco, Current Drugs Target 2009

SERUMPROTEOME

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Serum Samples

PATIENTS WERE PEOPLE DIAGNOSED WITH KNEE OA BETWEEN 57-95 YEARS OLD (ACR Criteria)

POOL 1

Grade II (K&L)10 patients

Control10 samples

Grade IV (K&L)10 patients

POOL 1,2,3,4,5

150 SAMPLES WERE USED IN THIS STUDY

Grade II (K&L)50 patients

Control50 samples

Grade IV (K&L)50 patients

Profile of Proteins in Serum

Disease biomarkers typically at low concentrations in plasma, making their detection difficult due to the presence of higher abundance proteins.

A practical and effective strategy to solve this problem is the removal of uninformative high abundance proteins to enhance the detection of low abundance proteins and penetrate deeper into the plasma proteome.

Depleted proteins by ProteoProteoPrep® 20 PlasmaImmunodepletion LC Column

Distribution of proteins in serum samples

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DEPLETION OF MOST ABUNDANT PROTEINS (TOP 20)

BIOINFORMATICS,PROTEIN PILOT

PROTEINS IDENTIFICATIONAND PEPTIDES QUANTFICATION USING MASS SPECTROMETER

POOLS OF SERUM SAMPLES(complex protein mixture)

WORKFLOW: Biomarkers in Serum

PROTEIN DIGESTION &PEPTIDES LABELING (with iTRAQ reagent)

114-CONTROL115-GRADE II116-GRADE IV

PEPTIDES SEPARATIONUSING LIQUID CROMATOGRAPHY

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Identified Proteins in Serum Pools

349 proteins were identified

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Quantified proteins in serum pools

PROTEIN NAME Pep (95%)

GradeII/Control PValue GradeIV/

Control PValue

Complement factor D 6 3.77 0.05 3.87 0.01

von Willebrand factor 21 5.55 0.00 5.30 0.00

Actin, cytoplasmic 1 8 0.69 0.00 0.72 0.00

C4b-binding protein alpha chain 55 0.51 0.42 0.37 0.07

Cartilage acidic protein 1 3 1.06 0.58 1.27 0.28

Sex hormone-binding globulin 8 0.41 0.20 0.86 0.78

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Joint Proteins

Seric Proteins

OsteoArthritis Biomarkers

Synovial Fluid and Cartilage

+

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SDS-PAGE nanoLC-MALDI-TOF/TOF Jesus Mateos

-SDS-PAGE in a 4-12% acrylamide Invitrogen pre-cast gel-Lane divided in 15 sections, excised and in-gel trypsin digested-Peptide extraction with 50%AcN, 0.1% TFA-Home made Poros® Desalting-nanoLC run with a 30 min gradient up to 50%AcN in a TEMPO nanoLC system (ABI)

-Hialuronidase treatment

-Immunodepletion using ProteoPrep 20 (Sigma) in a 1200 HPLC system (Agilent)

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Artro-CHIP of Proteins: Proteomic profile

OA profile Normal profile

Antibody Array(50 Proteins)

Statistic Analysis

Technical Validation

Antibody Array

Image of Microarray with labeled proteins Statistic Analysis

Validation of final microarrayARTRO-CHIP

Phase IPilot Microarray

(50 proteins)

Phase II(Screening and Confirmation)Microarray with 200 proteins

Phase III(Clinical Validation)

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Acknowledgments

Proteomics Unit

Patricia Fernandez-PuenteCristina Ruiz RomeroJesús MateosCarolina Fernández CostaValentina CalamiaBeatriz RochaLucia Lourido

Rheumatology Division

Carlos Fernández-LópezNatividad Oreiro-Villar

Mercedes Freire-Gonzalez

Orthopaedic Division

Alicia FrerireLourdes Sanjurjo

Dolores Velo

Colaboration

Manuel G. Fuentes