Analysis of Proteins Using DIGE and MALDI Mass Spectrometry - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 51

2.2. Spot Picking

1. 70% Denatured ethanol.
2. 7% Acetic acid.
3. Ettan spot picker (GE Healthcare, Piscataway, NJ, USA).

1. 25, 40, and 50 mM ammonium bicarbonate (ABC).
2. Acetonitrile (ACN).
3. 20

2.3. Manual In-Gel
Digestion

g/mL sequencing- or MS-grade trypsin in 25 mM ABC—
prepare immediately before use.
4. 0.1 and 3% trifl uoroacetic acid (TFA).

μ

1. 10% TFA.
2. Dissolving solution: 50% MeOH/0.1% TFA.
3. Matrix solution: 50% ACN/0.1% TFA/5 mM ammonium
citrate—prepare fresh before use.

2.4. MALDI-MS
Sample Preparation

2.5. MALDI-MS
Analysis

1. Peptide calibration standard.
2. 4800 MALDI-TOF/TOF mass spectrometer (AB Sciex, Foster
City, CA, USA).
3. GPS Explorer™ software (AB Sciex).
4. Mascot software (Matrix Science, London, UK).

3. Methods

1. Clean the Ettan spot picker thoroughly with 70% denatured
ethanol and rinse with pure water.
2. Use a 1.4-mm pick head (included with the instrument).
3. Prime the syringe at least 5 times to remove air bubbles and set
the syringe volume to 200

3.1. Spot Picking
Using a Commercial
Spot Picker

L.
4. Set up the gel in the gel tray and cover it slightly with pure
water (see Note 1).
5. Upload a pick list generated from the spot coordinates (see
Note 2).
6. Manually detect reference markers (see Note 1).
7. Set up a 96-well plate on the plate platform included with the
instrument.
8. Press “start” and let the picking robot perform spot picking
automatically.
9. Upon completion of the picking, remove the water from each
well, being careful not to remove the gel piece, and replace it
with 5

μ

L of 7% acetic acid.
10. Store the plate at 4°C until further sample preparation (see
Note 3).

μ