This document discusses matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and its role in proteomics. It provides an overview of two-dimensional gel electrophoresis (2-DE) and how it is used to separate thousands of proteins, including details on isoelectric focusing, immobilized pH gradients, sample preparation, electrophoresis, and silver staining. The core of proteomics is separating and analyzing proteins using techniques like 2-DE and mass spectrometry such as MALDI-TOF.

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