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Dissect the plasma protein markers for Parkinson’s disease

Dissect the plasma protein markers for Parkinson’s disease. Wang Vin-Chi, Lin Ching-Yu, and Chen Han-Min. About Parkinson’s disease. Who is suffered from Parkinson’s disease?. About Parkinson’s disease. 1. H istory A neuron system disease (James Parkinson, 1817)

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Dissect the plasma protein markers for Parkinson’s disease

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  1. Dissect the plasma protein markers for Parkinson’s disease Wang Vin-Chi, Lin Ching-Yu, and Chen Han-Min

  2. About Parkinson’s disease

  3. Who is suffered from Parkinson’s disease?

  4. About Parkinson’s disease 1. History • A neuron system disease (James Parkinson, 1817) • The second major neurodegenerative diseases in the world (after Alzheimer disease, AD) 2. Direct cause • The impairment of motor neuron cell in substantia nigra of midbrain.

  5. In the brain Normal image The neuronal death cause. • Those neuron cells produce dopamine as neuro-transmitter. A. Death of motor neuron cells • B. no dopamine • C. body motion dis-coordinate.

  6. About Parkinson’s disease 3. Major Symptoms • Generally, Parkinsons’ patients gradually lost the ability of writing, walking or showing facial countenance. Four clinical criteria • tremor • rigidity • bradykinesia • postural instability

  7. About Parkinson’s disease 4. Treatment From 1960, it is known that the Levodopa is effective to reduce symptom of 75% PD patients 5. The social cost on PD • 2,500 USD / year /patient • 56 hundred million / year / united state

  8. Diagnosis method • Base solely on doctor’s experience by evaluating the suspected patients for the four mentioned symptoms. • Disadvantages: • Not 100% accurate • Time consuming • Labor consuming • Can not be performed routinely

  9. Goal • To identify specific protein marker(s) from the blood of patients with Parkinson’s disease. • To develop a quick and convenient diagnosis method for Parkinson’s disease. Find in early days, treat in early Days.

  10. Approach To identify protein markers in blood for PD by proteomic approach

  11. Why proteins? Transporter for Genetic code Storage site for Genetic code Executor

  12. Protein: the true physiological executors

  13. Clinical diagnosis for blood samples

  14. Proteomic approach used What’s “proteomics” ? "The analysis of the entire protein complement expressed by a genome, or by a cell or tissue type.“ Two MOST applied techniques in proteomics: 1. 2-D electrophoresis Separation of complex protein mixtures 2. Mass spectrometry Identification of interested proteins.

  15. Proteomic work flow Healthy control Patient 1 st 2 nd Seperation Identification Digest to peptide fragment MS analysis

  16. What is 2-DE? • First dimension: • denaturing isoelectric focusing • separation according to the pI • 2. Second dimension: • SDS electrophoresis (SDS-PAGE) • Separation according to the MW Interested spot MS analysis Digest to peptide fragment

  17. MALDI-TOF Matrix Assisted Laser Desorption Ionization-Time Of Flight-MS Analysis Analyte molecules in matrix Vacuumlock Laser Vacuum system Ion detector Sampleplate Acceleration grids Drift tube Mass spectrum

  18. Annotation of protein by mass spectrometry Ionization Source Computing and Database Search Uninterpreted Data Mass Analyzer Detector Sample Introduction Postivie Protein ID Mass Spectrum Peak Assignment Raw mass data

  19. Our scenario

  20. Result

  21. Characteristics of samples

  22. Characteristics of samples (1-DE) Con Mr 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 PD Mr 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 97 kD 97 kD 97 kD 66 kD 66 kD 66 kD 45 kD 45 kD 45 kD 30 kD 30 kD 30 kD 21 kD 21 kD 21 kD Mr 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

  23. Depletion of abundant serum proteins from plasma samples (Original sample pools) CBB W.B. Mr Con PD Con PD anti transferrin anti a1-antitrypsin anti IgG kappa light chain CBB CBB Con PD Con PD 97 kD 66 kD 45 kD 30 kD 21 kD albumin 1.5M NaCl IgG heavy chain 0.1M Glycine IgG light chain CBB Con PD (Eluted fractions) (Flow through)

  24. 2-DE separation (median format) 97 kD 66 kD 45 kD 30 kD 21 kD 14 kD Con PD pH 7 pH 4 pH 7 pH 4 Mr Mr

  25. 2-DE separation (large format) pH 7 pH 4 Mr 97 kD 66 kD 45 kD 30 kD 21 kD 14 kD 1 2 PD

  26. MALDI Q-TOF annotation Serum amyloid P component 1

  27. MALDI Q-TOF annotation 2 IgG kappa light chain 1

  28. Immunological validation (1-DE WB) Fold Mr Con PD Con PD 220 kD 97 kD 3.3X 66 kD 3.9X 45 kD 30 kD 5.9X 20.1 kD 14.3 kD 10.4X

  29. Immunological validation (2-DE WB) 97 kD 66 kD 45 kD 30 kD 21 kD Con PD pH 7 pH 4 pH 7 pH 4 Mr Mr a2-macroglobulin ceruloplasmin a1-B glycoprotein transferrin albumin Hemopexin IgA a chain a1-antitrypsin Transferrin fragment CD5 antigen like Leucine rich a2-glycoprotein Haptoglobin b Zinc finger protein APO A-IV precursor Transthyretin + retinol binding protein IgG light chain APO A-I Haptoglobin a2 Transthyretin

  30. Immunological validation (2-DE WB) 220 kD 97 kD 66 kD 45 kD 30 kD 21 kD 14 kD Con PD pH 4 pH 7 pH 4 pH 7 Mr Mr

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