1. Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass spectrometry for protein identification
2-Dimensional Gel Electrophoresis
MALDI-TOF Mass Spectrometry
Speaker: Dr. J. S. Yu
Date: 3/21/2002

2. The age of X-omics and biotechnology: Genomics: Human genome project
Transcriptomics: cDNA microarray
Proteomics:
Development and involvement of mass spectrometry
Celera Genomics Inc.
cDNA microarray
Tandem mass spectrometer (MS/MS)
MALDI-TOF MS

3. Proteomics solution
IEF
SDS-PAGE

4. 2-Dimension Electrophoresis (2-DE) for Protein Separation
The core technology of proteomics is 2-DE: At present, there is no other technique which is capable of resolving thousands of proteins in one separation procedure.
Speaker: C. C. Wu
Date: 31/10/2001

5. Isoelectric point (pI):
Isoelectric point is the pH of a solution at which the net charge of protein is zero. In electrophoresis there is no motion of the particles in an electric field at the isoelectric point.
NH3+
COOH
pH < pI
Positive charge
NH3+
COO-
pH = pI
NH2
COO-
pH > pI
Negative charge
Net charge pH
Isoelectric point

6. General principle and protocol of 2-Dimension Electrophoresis
Ampholytes
sample
Isoelectric focusing
(1st dimension) pH pH
polyacrylamide
2nd dimension
SDS-PAGE MW
pH gradient

7. Traditional Equipment for Isoelectric focusing (IEF):
Ampholytes
polyacrylamide
Cathode (-) electrode
solution
Anode (+) electrode solution

8. Traditional 2-Dimensional Electrophoresis
Disadvantage: cathodic drift
Cathode (-) electrode solution
pH pH pH 5
polyacrylamide
Ampholyte
pH pH pH 3
Time
Anode (+) electrode solution

9. Immobilized pH Gradient (IPG)
Acrylamide monomer
Acidic buffering group:
COO-
CH2 = CH-C-NH-R O
Basic buffering group:
NH3+
Polyacrylamide gel

10. Production of Immobilized pH Gradient (IPG) strip
Gradient maker
acidic
basic A D
plastic support
film B E C F
pH 3
pH 10

11. Equipment for Isoelectric focusing (IEF):
IPGphor (IEF System)
Amersham Pharmacia Biotech Inc.
Protein IEF Cell
Bio-Rad Laboratories

12. Sample preparation Lysis solution: 8M Urea 4% NP-40 or CHAPS
40mM Tris base
Cell line
Lysis solution
Sonication
vacuum
Lysis solution
Centrifugation
Measurement of [protein]
2-DE sample

13. IPG strip rehydration and sample loading
Rehydration solution
2-DE sample
Rehydration solution:
8M Urea
2% NP-40 or CHAPS
2% IPG buffer (Ampholyte)
0.28% DTT
Trace Bromophenol blue
IPG strip holder
Position the
IPG strip

14. IPG strip rehydration and sample loading
Strip holder
Anode (+)
electrode
Cathode (-)
electrode
30 voltage 12hr

15. First dimension: Isoelectric focusing
1. Place electrode pads (?)
V step-n-hold hr
V step-n-hold hr
V gradient vhr
V gradient (?) vhr
Holder cover
IPG strip
Electrode
pads
Time
Voltage

16. Second dimension: SDS-PAGE
SDS equilibration
SDS-PAGE
SDS
SDS equilibration buffer
50 mM Tris-HCl
6 M Urea
30% Glycerol
2% SDS
Trace Bromophenol
IPG strip
SDS-PAGE
0.5% agarose
in running buffer
Marker in paper

17. Protocol of silver stain:
50% methanol
25% acetic acid
4hr
ddH2O
30 sec
ddH2O x 3 times
30min/time
3% Na2CO3
0.0185% formaldehyde
0.004% DTT solution
30min
2.3M citric acid
0.1% AgNO3
30min
5% acetic acid
25% methanol

18. 2-DE separation of soluble E. coli proteins