PROTEOMICS APPROACH FOR THE SEARCH OF OA...
Transcript of PROTEOMICS APPROACH FOR THE SEARCH OF OA...
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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