In-Solution Digestion Protocols

In-solution digestion protocols

Protocol A:

1. Make sure proteins are in an appropriate buffer (20mM Tris-HCl, or 20mM Ammonium Bicarbonate, pH 7.5-8). Protein concentration should be around 1 mg/ml.

2. To 100 ml of protein solution, add 1 ml of 10% SDS.

3. Add 2 ml of 100 mM TCEP. Incubate at 56°C for 20min (or longer).

4. Add 10 ml 200 mM IAA (fresh made) and incubate at room temperature for 15 min (or longer) in the dark.

5. Add trypsin to a final concentration of 10 ng/ml. Incubate at 37°C overnight.

6. Add 10´ SCX Buffer A (total salts should be less than 15 mM). Check to make sure pH is between 2.5 and 3.3 (using pH paper). If not, adjust with 10% Phosphoric acid. (If the initial sample contains salts, the above protocols can still apply, as long as the final pH is around 7.5-8. However, it should be diluted with more SCX buffer A to make sure the final salts are less than 15 mM. Alternatively, a desalting step with Sep-Pak or equivalent can be performed).

7. Perform SCX to remove SDS and other contaminants.

8. Perform Sep-Pak desalting or equivalent.

9. Dry the sample with lyophilizer and SpeedVac.

10. Reconstitute the sample in 10-20 ml of reversed-phase buffer A. Spin at full speed in a desktop centrifuge for 10 min. Load the supernatant into an LC sample vial (make sure there’s no bubble at the bottom of the vial).

Protocol B:

1. Make sure proteins are in an appropriate buffer (20mM Tris-HCl, or 20mM Ammonium Bicarbonate, pH 7.5-8). Protein concentration should be around 1 mg/ml.

2. To 100 ml of protein solution, add 2 ml of 1% ProteaseMax.

3. Add 2 ml of 100 mM TCEP. Incubate at 56°C for 20min.

4. Add 10 ml 200 mM IAA (fresh made) and incubate at room temperature for 15 min in the dark.

5. Add trypsin to a final concentration of 10 ng/ml and add another 1 ml of 1% ProteaseMax. Incubate at 37°C for 3 hrs or overnight.

6. Quick spin to collect the sample. Add TFA to a final of 0.5% and incubate at room temperature for 5 min.

7. Spin at full speed in a desktop centrifuge for 10 min. keep the supernatant.

8. Perform Sep-Pak desalting or equivalent.

9. Dry the sample with lyophilizer and SpeedVac.

Note: 1% ProteaseMax stock solution (in 50 mM Ammonium Bicarbonate) has to be kept on ice while in use, and kept at -20°C. It should not be freeze-thawed for more than 5 times.