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?

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

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

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

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

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

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

To identify protein markers in blood for PD by proteomic approach

Approach

Storage site forGenetic code

Transporter forGenetic code

Executor

Why proteins?

Protein: the true physiological executors

Clinical diagnosis for blood samples

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

Healthy controlPatient

Digest to peptide fragment MS analysis

1 st

2 ndSeperation Identification

Proteomic work flow

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?

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

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

Our scenario

Result

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

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)

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

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

2-DE separation (large format)

1

2

97 kD

66 kD

45 kD

30 kD

21 kD

14 kD

MrpH 4 pH 7

PD

AYSLFSYNTQGR

AYSLFSYNTQGRDNELLVYK DNELLVYKER

VGEYSLYIGR

IVLGQEQDSYGGKFDR

MALDI Q-TOF annotation

1

Serum amyloid P component

K.SGTASVVCLLNNFYPR.E

K.VYACEVTHQGLSSPVTK.S

R.TVAAPSVFIFPPSDEQLK.S

MALDI Q-TOF annotation

1

IgG kappa light chain

2

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)

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

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)