In-Gel Digestion Proteomics Protocol for Peptide Mass Fingerprinting and Mass Spectrometry

Biochemistry, Molecular Biology, and Cell Biology Protocols >> In-gel Digestion Proteomics Protocol for Peptide Mass Fingerprinting and Mass Spectrometry

0.1M Ammonium Bicarbonate should be made fresh for every set of digestions (can be stored for one day in the fridge, no need to filter)

Washing
1. Place some water into maxymum recovery tubes.
2. Excise chunks of your Coomassie-stained band using a P1000 pipet tip by punching down, and then sucking up with the pipet.
3. Can repeat for several chunks before pipeting the chunks into the maxymum recovery tubes.
4. Wash the gel pieces by placing 100uL of 0.1M ammonium bicarbonate and sonicating for 15 minutes (no need to heat).
5. Spin down the liquid.
6. Add an equal volume (100uL) of acetonitrile and sonicate for another 15 minutes
7. Spin down the liquid.
8. Remove all the liquid and repeat washing step if necessary
9. After each washing step, put the tube against a piece of white paper to check if all gel pieces are white
10. If they are white, no need to do anymore washing.
11. Add acetonitrile and shrink.

Reduction and Alkylation
1. Add 50uL10mM DTT in 0.1M ammonium bicarbonate 56 degrees C, 30 minutes, shaking.
2. Spin down, remove, and add acetonitrile to shrink.
3. Add 50uL 55mM iodoacetamide in 0.1M ammonium bicarbonate and incubate 20minutes in dark; flick it every now and then.
4. Spin down.
5. Wash with 0.1M ammonium bicarbonate (150-200uL). Repeat if desired.
6. Spin down.
7. Shrink with acetonitrile.

Digestion
1. Rehydrate in digestion buffer.
Composition of digestion buffer: 50uL water, 50uL 0.1M ammonium bicarbonate, 15uL 0.1ug/uL porcine trypsin, 5uL 1% calcium chloride)
2. Spin down and remove after 30 minutes on ice.
3. Add more of digestion buffer without the trypsin.
4. Incubate overnight 37 degrees C with shaking.

Extraction
1. The next day, transfer digestion to new maxymum recovery tube.
2. Add 20uL 0.1M ammonium bicarbonate and sonicate for 15 minutes
3. Spin down.
4. Add 40uL acetonitrile and sonciate for another 15 minutes.
5. Remove and combine into the digestion tube.
6. Add 50uL formic acid and sonicate for 15 minutes.
7. Add 100uL acetonitrile and sonicate for another 15 minutes.
8. Combine.
9. Divide into two or more tubes if necessary, freeze in liquid nitrogen, lyophilize and freeze in –20 degrees C.