In Gel Digestion of Proteins for MALDI-MS

Gel fragment preparation

Excise protein bands. Cut each into 1 mm pieces. Place into a siliconized tube. Also cut out a gel piece from a protein-free region as control.
Wash gel pieces with >10 volumes of Millipore water [~200ul] for 30 seconds, to wash out acetic acid.

Destaining

For Coomassie Blue staining, destain two times for 10 minutes or until colorless with 200ul 100 mM (NH₄)HCO₃/50% Methanol (discard supernatants). Dehydrate for 5 minutes with 200ul 25 mM (NH₄)HCO₃/50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile. Remove the solutions and discard. The gel pieces shrink and become white.
For Non-Destructive Silver staining, destain twice for 5 minutes or until colorless with 200ul of freshly prepared 1:1 solution of 100mM Sodium Thiosulfate [Na₂S₂O₃] and 30mM Potassium Ferricyanide [K₃Fe(CN)₆]. Stop the reaction and wash out silver ions twice for 5 minutes with 500ul of Millipore water. Dehydrate for 5 minutes with 200ul 25mM (NH₄)HCO₃/50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile. The gel pieces shrink and become white.
For Sypro Ruby staining, no destaining necessary, Dehydrate for 5 minutes with 200ul 25mM (NH₄)HCO₃/50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile. Remove the solutions and discard. The gel pieces shrink and become white.
Dry gel particles for 5 minutes in a vacuum centrifuge.

Reduction and Alkylation

Rehydrate gel pieces in 50ul of freshly prepared 25mM Dithiothreitol (in 25mM (NH₄)HCO₃). Reduce the proteins for 20 minutes at 56°C.
Cool the gel to room temperature, pipet off any residual liquid and add 50ul of freshly prepared 55mM Iodoacetamide (in 25mM (NH₄)HCO₃). Alkylate the proteins for 20 minutes at room temperature in the dark.
Pipet off the liquid and wash gel pieces with > 20 volumes of Millipore water [~400ul] for 30 seconds to remove any residual Iodoacetamide. Dehydrate gel pieces for 5 minutes with 200ul 25mM (NH₄)HCO₃/50% Acetonitrile then once more for 30 seconds in 100% Acetonitrile.
Dry gel particles for 5 minutes in a vacuum centrifuge.

Trypsin Digestion

Rehydrate gel pieces for 5 minutes at room temperature in 20ul [20ng/ul] Trypsin (Promega Sequence Grade Modified) in 25 mM (NH₄)HCO₃ / 3%Acetonitrile [ pH =~8.5].
Overlay the rehydrated gel particles with a minimum amount of 25mM (NH₄)HCO₃ to keep them immersed throughout the digestion. Incubate 16-24 hours at 37C.

Peptide Recovery

Extract digested peptides with 50ul Millipore water/0.1% TFA by vortexing 15 minutes at room temperature (max speed). Transfer solution to a new siliconized microcentrifuge tube.
Perform an additional extraction with 80ul of 70%ACN/25% H2O/5%TFA.
Dry peptide solution completely in a vacuum centrifuge ( 1-2 hours).
Reconstitute peptides in 30ul of Millipore water/0.1%TFA by incubating for 5 minutes at room temperature with intermittent vortexing.
Perform ZipTip-C18 column (Millipore) clean-up before spotting onto MALDI plate.

References