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. 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. About Parkinson’s disease

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

6. 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 About Parkinson’s disease

7. 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 About Parkinson’s disease

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. 1.To identify specific protein marker(s) from the blood of patients with Parkinson’s disease. 2.To develop a quick and convenient diagnosis method for Parkinson’s disease. Find in early days, treat in early Days. Goal

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

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

12. Protein: the true physiological executors

13. Clinical diagnosis for blood samples

14. 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. Proteomic approach used

15. Healthy control Patient Digest to peptide fragmentMS analysis 1 st 2 nd SeperationIdentification Proteomic work flow

16. Digest to peptide fragment MS analysis 1.First dimension: denaturing isoelectric focusing separation according to the pI 2. Second dimension: SDS electrophoresis (SDS-PAGE) Separation according to the MW Interested spot What is 2-DE?

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

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

19. Our scenario

20. Result

21. Characteristics of samples

22. Con Mr12345678910111213141516 97 kD 66 kD 45 kD 30 kD 21 kD PD Mr 97 kD 66 kD 45 kD 30 kD 21 kD 12345678910111213141516 1718 Mr 97 kD 66 kD 45 kD 30 kD 21 kD 19202122232425262728293031323334 3536 Characteristics of samples (1-DE)

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

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

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

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

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

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

29. Immunological validation (2-DE WB) Con Mr pH 4 pH 7 PD Mr pH 4 pH 7 transferrin  2-macroglobulin  1-B glycoprotein ceruloplasmin albumin IgA  chain Transferrin fragment  1-antitrypsin Leucine rich  2-glycoprotein CD5 antigen like APO A-IV precursor APO A-I Haptoglobin  Transthyretin + retinol binding protein IgG light chain Zinc finger protein Hemopexin Haptoglobin  2 97 kD 66 kD 45 kD 30 kD 21 kD Transthyretin

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