Reagents
1. Lysis buffer: 8M urea, 50 mM Tris-HCl (or 50 mM ammonium bicarbonate), pH 8. Add 1*EDTA-free protease inhibitor (Roche Diagnostics) before use, add HDAC or phosphatase inhibitors if needed.
2. DTT: dithiothritol (4°C), to make 1 M stock, 15.4 mg/100 µL water.
3. IAA: iodoacetamide (4°C), to make 1 M stock, 18.5 mg/100 µL water.
4. Loading buffer: 80% Acetonitrile, 5% TFA and 1 M Glycolic acid (76 mg/mL)
5. Washing buffer 1: 80% Acetonitrile, 1% TFA
6. Washing buffer 2: 20% Acetonitrile, 0.2% TFA
7. Elution buffer: 40 µL Ammonia solution (28%) in 980 µL H2O, pH 11,3
Cell lysis
1. Harvest cells with a scraper and wash three times with PBS.
2. Suspend the cell pellet in lysis buffer in a ration of ~3:1 to 5:1 (buffer to pellet). Pipette up and down to resuspend pellet. Kept on ice for 10 min.
3. Sonicate on ice 30 seconds on, 60 seconds off. Repeat 3 to 5 times total.
4. Spin max speed on centrifuge (use the one in cold room) to pellet debris for 10 min.
5. Keep supernatant and determine protein concentration by Bradford or BCA assay.
Reduction and alkylation proteins
1. Add 1M DTT stock solution to a final concentration of 5 mM, and incubate the sample for 1 hour at 56°C.
2. Add 1M IAA stock solution to a final concentration of 10 mM, and incubate the sample for 45 min at room temperature in the dark.
3. Quench the reaction by adding DTT stock solution up to a final concentration 10 mM, and incubate for 10 min at room temperature.
Digestion
1. Dilute with 50 mM Tris-HCl (or 50 mM ammonium bicarbonate), pH 8, until urea concentration is lower than 2.0 M (1.5 M is fine).
2. Add trypsin in a ratio 1:50 (enzyme: protein), and incubate overnight at 37°C. If doing large scale proteome digestion (more than 2 mg), a ratio of 1:100 can be used.
3. Stop the digest by acidifying the sample to a pH of 2 by adding TFA.
4. Dry down sample by SpeedVac and stored at -20°C.
5. Stage-tip before inject into LC-MS.
