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1. Matrix-Assisted Laser Desorption Ionization Time-of-
Flight (MALDI-TOF) Mass spectrometry for protein
identification
• 2-Dimensional Gel Electrophoresis
• MALDI-TOF Mass Spectrometry
M.PRASAD NAIDU
Msc Medical Biochemistry,
Ph.D Research scholar.

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

3. Proteomics solution
IEF
SDS-PAGE

4. 2-Dimension Electrophoresis (2-DE) for Protein Separation
Speaker: C. C. Wu
Date: 31/10/2001
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.

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.
Netcharge
-3
-2
-1
0
1
2
3
2 3 4 5 6 7 8 9 10 11
pH
Isoelectric point
NH3
+
COOH
NH3
+
COOH
pH < pI
Positive charge
NH3
+
COO-
NH3
+
COO-
pH = pI
NH2
COO-
NH2
COO-
pH > pI
Negative charge

6. sample
pH 9 -
pH 3 +
Isoelectric focusing
(1st dimension)
General principle and protocol of 2-Dimension Electrophoresis
MW
pH gradient
SDS-PAGE
Ampholytes
polyacrylamide
2nd dimension

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

8. Traditional 2-Dimensional Electrophoresis
Anode (+) electrode solution
Cathode (-) electrode solution
Disadvantage: cathodic drift
Ampholyte
polyacrylamide
pH 3 pH 3 pH 3
pH 9 pH 7 pH 5
Time

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

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

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

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

13. IPG strip rehydration and sample loading
2-DE sample Rehydration
solution
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
Cathode (-)
electrode
Anode (+)
electrode
30 voltage 12hr

15. First dimension: Isoelectric focusing
1. Place electrode pads (?)
2. 200 V step-n-hold 1.5hr
3. 500 V step-n-hold 1.5hr
4. 1000 V gradient 1500vhr
5. 8000 V gradient (?) 36000vhr
Time
Voltage
Holder cover
IPG strip
Electrode
Electrode
pads

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

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

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