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 g/l.
2. To 100 l of protein solution, add 1 l of 10% SDS.
3. Add 2 l of 100 mM TCEP. Incubate at 56C for 20min (or longer).
4. Add 10 l 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/l. 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 l 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 g/l.
2. To 100 l of protein solution, add 2 l of 1% ProteaseMax.
3. Add 2 l of 100 mM TCEP. Incubate at 56C for 20min.
4. Add 10 l 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/l and add another 1 l 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.
10. Reconstitute the sample in 10-20 l 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).
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.