Dissect the plasma protein markers for Parkinson’s disease
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(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 t he 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-transmit ter. 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 abilit y 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 re duce 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 evalua ting the suspected patients for the four menti oned symptoms. Disadvantages: Not 100% accurate Time consuming Labor consuming Can not be performed routinely.
(9) Goal. 1. To identify specific protein marker(s) from the b lood of patients with Parkinson’s disease. 2. To develop a quick and convenient diagnosis m ethod 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?. Storage site for Genetic code. Transporter 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. 1 st. Patient. Healthy control. 2 nd Seperation. Digest to peptide fragment. Identification. MS analysis.
(16) What is 2-DE?. 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. Digest to peptide fragment. MS analysis.
(17) Vacuum lock. MALDI-TOF Matrix Assisted Laser Desorption Ionization-Time Of Flight-MS Analysis. r e s a L Vacuum system. Sample Analyte Acceleration plate molecules grids in matrix. Drift tube Mass spectrum. Ion detector.
(18) Annotation of protein by mass spectrometry Sample Introduction. Postivie Protein ID. Computing and Database Search Ionization Source Uninterpreted Data Mass Spectrum Peak Assignment. Mass Analyzer Detector. Raw mass data.
(19) Our scenario.
(20) Result.
(21) Characteristics of samples. Group. Con. PD. Clinical diagnosed with PD. no. yes. Female. 9. 16. Male. 7. 20. 56-86. 56-85. 67.9. 76.8. Age span (Min.-Max.) Plasma protein Conc. (mg/mL).
(22) Characteristics of samples (1-DE). Con Mr 1 97 kD 66 kD 45 kD. 2 3. 4 5 6 7. 8. 9. PD Mr 1. 10 11 12 13 14 15 16. 2 3. 4 5 6 7. 8. 9. 10 11 12 13 14 15 16 17 18. 97 kD 66 kD 45 kD. 30 kD. 30 kD. 21 kD. 21 kD. Mr 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 97 kD 66 kD 45 kD 30 kD 21 kD.
(23) Depletion of abundant serum proteins from plasma samples (Original sample pools) Mr. 97 kD 66 kD. CBB. W.B.. CBB. CBB. Con PD. Con PD. Con PD. Con PD. anti transferrin albumin. anti a1-antitrypsin. 1.5M NaCl. 45 kD. 0.1M Glycine. IgG heavy chain. 30 kD IgG light chain. anti IgG kappa light chain. 21 kD. CBB Con PD. (Flow through). (Eluted fractions).
(24) 2-DE separation (median format) Con Mr. 97 kD 66 kD 45 kD. 30 kD. 21 kD 14 kD. pH 4. PD pH 7. Mr. pH 4. pH 7.
(25) 2-DE separation (large format) PD Mr. pH 7. pH 4. 97 kD 66 kD 45 kD 30 kD. 21 kD 14 kD. 1 2.
(26) MALDI Q-TOF annotation Serum amyloid P component VGEYSLYIGR 1. AYSLFSYNTQGR IVLGQEQDSYGGKFDR. DNELLVYKER. AYSLFSYNTQGRDNELLVYK.
(27) MALDI Q-TOF annotation IgG kappa light chain K.SGTASVVCLLNNFYPR.E 1. R.TVAAPSVFIFPPSDEQLK.S K.VYACEVTHQGLSSPVTK.S. 2.
(28) Immunological validation (1-DE WB). Mr. Con PD. Fold Con. PD. 220 kD 97 kD. 3.3X. 66 kD 45 kD. 3.9X. 30 kD 5.9X 20.1 kD 14.3 kD. 10.4X.
(29) Immunological validation (2-DE WB). Con Mr. PD pH 7. pH 4 ceruloplasmin. 2-macroglobulin. 97 kD 1-B glycoprotein. 66 kD. transferrin. albumin Hemopexin. IgA chain 1-antitrypsin. 45 kD. Leucine rich 2-glycoprotein. Transferrin fragment CD5 antigen like. Haptoglobin Zinc finger protein. APO A-IV precursor Transthyretin + retinol binding protein. 30 kD. IgG light chain APO A-I. 21 kD. Haptoglobin 2. Transthyretin. Mr. pH 4. pH 7.
(30) Immunological validation (2-DE WB) Con Mr. 220 kD 97 kD 66 kD 45 kD 30 kD. 21 kD 14 kD. pH 4. PD pH 7. Mr. pH 4. pH 7.
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