dissect the plasma protein markers for parkinson’s disease wang vin-chi, lin ching-yu, and chen...
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Dissect the plasma protein markers for Parkinson’s disease
Wang Vin-Chi, Lin Ching-Yu, and Chen Han-Min
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About Parkinson’s disease
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Who is suffered from Parkinson’s disease?
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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.
About Parkinson’s disease
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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
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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
About Parkinson’s disease
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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
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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
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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
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To identify protein markers in blood for PD by proteomic approach
Approach
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Storage site forGenetic code
Transporter forGenetic code
Executor
Why proteins?
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Protein: the true physiological executors
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Clinical diagnosis for blood samples
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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
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Healthy controlPatient
Digest to peptide fragment MS analysis
1 st
2 ndSeperation Identification
Proteomic work flow
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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?
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Laser
Sampleplate
Analytemolecules in matrix
Accelerationgrids
Drift tube Ion detector
Mass spectrum
Vacuum system
Vacuumlock
MALDI-TOF Matrix Assisted Laser Desorption Ionization-Time Of Flight-MSAnalysis
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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
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Our scenario
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Result
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Group Con PD
Clinical diagnosed with PD no yes
Female 9 16
Male 7 20
Age span (Min.-Max.) 56-86 56-85
Plasma protein Conc. (mg/mL) 67.9 76.8
Characteristics of samples
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Con
Mr 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
97 kD
66 kD
45 kD
30 kD
21 kD
PD
Mr
97 kD
66 kD
45 kD
30 kD
21 kD
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Mr
97 kD
66 kD
45 kD
30 kD
21 kD
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Characteristics of samples (1-DE)
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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 chain0.1M Glycine
albumin
(Eluted fractions)
Con PD
CBB
Con PD
CBB
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2-DE separation (median format)
Con
MrpH 4 pH 7
PD
MrpH 4 pH 7
97 kD
66 kD
45 kD
30 kD
21 kD
14 kD
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2-DE separation (large format)
1
2
97 kD
66 kD
45 kD
30 kD
21 kD
14 kD
MrpH 4 pH 7
PD
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AYSLFSYNTQGR
AYSLFSYNTQGRDNELLVYK DNELLVYKER
VGEYSLYIGR
IVLGQEQDSYGGKFDR
MALDI Q-TOF annotation
1
Serum amyloid P component
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K.SGTASVVCLLNNFYPR.E
K.VYACEVTHQGLSSPVTK.S
R.TVAAPSVFIFPPSDEQLK.S
MALDI Q-TOF annotation
1
IgG kappa light chain
2
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45 kD
30 kD
20.1 kD
66 kD
97 kD
220 kD
14.3 kD
Con PDMrFold
3.3X
3.9X
5.9X
10.4X
Con PD
Immunological validation (1-DE WB)
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Immunological validation (2-DE WB)
Con
MrpH 4 pH 7
PD
MrpH 4 pH 7
transferrin
2-macroglobulin
1-B glycoprotein
ceruloplasmin
albumin
IgA chain
Transferrinfragment
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
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97 kD
66 kD
45 kD
30 kD
21 kD
14 kD
220 kD
Con
MrpH 4 pH 7
PD
MrpH 4 pH 7
Immunological validation (2-DE WB)