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 NAIDUMsc Medical Biochemistry,Ph.D Research scholar.

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.

Proteomics solution

IEF

SD

S-P

AG

E

2-Dimension Electrophoresis (2-DE) for Protein Separation

Speaker: C. C. WuDate: 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.

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.

Net

cha

rge

-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 < pIPositive charge

NH3+

COO-

NH3+

COO-

pH = pI

NH2

COO-

NH2

COO-

pH > pINegative charge

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

Traditional Equipment for Isoelectric focusing (IEF):

Ampholytespolyacrylamide

Cathode (-) electrode solution

Anode (+) electrode solution

Traditional 2-Dimensional Electrophoresis

Anode (+) electrode solution

Cathode (-) electrode solution

Disadvantage: cathodic driftA

mph

olyt

epo

lyac

ryla

mid

e

pH 3 pH 3 pH 3

pH 9 pH 7 pH 5

Time

Immobilized pH Gradient (IPG)

Polyacrylamide gel

Acidic buffering group:

Basic buffering group:CH2 = CH-C-NH-R

O

COO-

NH3+

Acrylamide monomer

Gradient maker

plastic support film

Production of Immobilized pH Gradient (IPG) strip

A

C

B

F

E

Dacid

ic

basi

c

pH 3

pH 10

IPGphor (IEF System)Amersham Pharmacia Biotech Inc.

Protein IEF CellBio-Rad Laboratories

Equipment for Isoelectric focusing (IEF):

Lysis solution:8M Urea4% NP-40 or CHAPS40mM Tris base

Sample preparation

Cell line

Lysis solution

Sonication

vacuum

Lysis solution

Centrifugation

Measurement of [protein]

2-DE sample

IPG strip rehydration and sample loading

2-DE sample Rehydration solution

Rehydration solution:8M Urea2% NP-40 or CHAPS2% IPG buffer (Ampholyte)0.28% DTTTrace Bromophenol blue

IPG strip holder

Position the

IPG strip

IPG strip rehydration and sample loading

Strip holder

Cathode (-) electrode

Anode (+) electrode

30 voltage 12hr

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

Vol

tage

Holder cover

IPG strip

Electrode

Electrode pads

Second dimension: SDS-PAGE• SDS equilibration• SDS-PAGE

SDS equilibration buffer50 mM Tris-HCl6 M Urea30% Glycerol2% SDSTrace Bromophenol

SD

S

SDS-PAGE SDS-PAGE

0.5% agarose in running buffer

SDS-PAGE

Marker in paper

IPG strip

Protocol of silver stain:

50% methanol25% acetic acid4hr

ddH2O x 3 times

30min/time

0.004% DTT solution30min

0.1% AgNO3

30min

ddH2O

30 sec

3% Na2CO3

0.0185% formaldehyde

2.3M citric acid

5% acetic acid25% methanol

2-DE separation of soluble E. coli proteins