Remove the gel piece from the bottom of the tube using a small spatula. Chop up the cone-shaped acrylamide using a scalpel blade. If you can't get the cone out of the tube, use the spatula to chop it into pieces in situ, but don't macerate it!
Cover the gel pieces with destain solution for incubate for 10 minutes with occasional mixing.
Spin down and remove the destain.
Cover the pieces with 6 M urea (~500 ul) and incubate for 10 minutes with occasional mixing.
Add acetonitrile (~50% of urea volume) and vortex. Remove supernatant. Repeat if high concentrations of SDS or other contaminants are present.
Rehydrate in 100 mM Ammonium Bicarbonate (~200ul) and incubate for 10 minutes with occasional mixing.
Add an equal volume of acetonitrile. Vortex and spin down. Remove supernatant.
Add 100% acetonitrile and allow to dehydrate completely.
Remove acetonitrile and air dry.
Add ~50 ul of 5 ng/ul trypsin (in 50 mM AB). Let sit on ice for 10 min. Add more trypsin solution if the gel pieces aren't fully rehydrated.
Add an equal volume of 50mM AB and incubate overnight at 37^o C.
In the morning, add 50 ul of acetonitrile to the sample and collect the liquid into a clean tube.
Add ~100 ul of extraction solution, vortex and incubate 10 min. Spin down and add the supernatant to the same sample tube.
Add ~100 ul of Extraction solution: Acetonitrile (50:50). Vortex and incubate 10 min. Spin down and add the supernatant to the same sample tube.
Dry down and resuspend in solvent A (.1% FA in water)
Desalt

Notes on Data Analysis

GASP will yield peptides with propionamide on lysine and the N-termini. These should be considered as variable modifications.

Cysteines will also be modified to PAM-Cys with the propionamide. This is a fixed modification.

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