General proteomics FASP (Filter-Aided Sample Preparation)

Prepare fresh buffers in LC-MS water:

Exchange buffer: 8M urea, 0.2% (w/v) deoxycholate, 1M ammonium bicarbonate (pH 8)

Digestion buffer: 0.2% (w/v) deoxycholate, 50 mM ammonium bicarbonate (pH 8)

Trypsin dissolution/peptide recovery buffer: 50 mM ammonium bicarbonate (pH 8)

Dispense 25-50μL (depending on concentration) reduced, alkylated protein extract to filter unit.

Fill filter units with 200 μL exchange buffer, mix with pipet.

Spin at 14,000g for 10 min, discard filtrate.

Repeat steps 3 and 4 three more times.

Wash filter unit with 200 μL digestion buffer, and spin at 14,000g for 10 min. Do this a total of 2 times.

Transfer filter unit to passivated collection tube.

Add 110 μL digestion buffer and desired amount of trypsin (~1:50 trypsin/protein), dissolved in 50 mM ammonium bicarbonate, keeping trypsin on ice or at 4° C. Incubate at 37° C overnight. Parafilming tubes is recommended to prevent evaporation.

Remove parafilm if applicable and centrifuge tubes at 14,000g for 10 min. Do not discard filtrate!

Add 50 μL of peptide recovery buffer to filters, spin at 14,000g for 10 min. Do this step a total of 2 times.

Transfer the filtrate to a LoBind tube.

Add 900μL ethyl acetate and 2.5 μL TFA to filtrate, vortex. Precipitate may be visible.

Sonicate in a bath sonicator for 10s, and centrifuge at 16,000g for 10 min.

Carefully remove upper organic layer with a pipet without disturbing phase boundary.

Repeat steps 12, 13 and 14 two more times (Note: Only add 900μL ethyl acetate, no more TFA)

Place sample uncovered in Thermomixer and dry at 60° C for 5-10 minutes until ethyl acetate is gone.

Freeze sample solid at -80° C, Centrivap to dryness.