Tryptic digestion directly in polyacrylamide gel slices
This protocol is designed for the direct digestion of proteins in Coomassie stained polyacrylamide gels. Direct digestion in the gel band eliminates transfer of the protein of interest to a membrane and allows Coomassie staining to be used. The absence of detergents is an additional advantage of this protocol because it allows direct analysis of the digest by capillary LC-tandem mass spectrometry without off-line HPLC fractionation. Our experiments have found the limit of detection for this protocol to be approximately 1 pmol of protein in the gel. Because this limit of detection is similar to the limit of detection of Coomassie staining, we believe that any gel band that is reasonably stained by Coomassie blue represents sufficient protein for sequence analysis by capillary column LC-electrospray mass spectrometry. The protocol is based on the published method of Mann for the digestion of silver stained gels for analysis by nanospray mass spectrometry. We have used this protocol on silver stained spots although keratin contamination commonly interferes.
Reducing trypsin concentration below 10 ng/µl may reduce sequence coverage (http://www.asms.org/Proceedings/1143.pdf).
A. Shevchenko, M. Wilm, O. Vorm, and M. Mann. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Analytical Chemistry 68:850-858 (1996).
Reagents (All reagents prepared fresh)
1. Destain: 50% methanol/5% acetic acid in water.
2. 100 mM ammonium bicarb: 100 mM ammonium bicarbonate in distilled water: 0.158 g/20 ml.
3. 50 mM ammonium bicarb: 50 mM ammonium bicarbonate in distilled water.
5. 10 mM DTT: 1.5 mg/mL in 100 mM ammonium bicarb.
6. 50 mM iodoacetamide: 10 mg/mL in 100 mM ammonium bicarb.
7. Trypsin solution (on ice): 20 ng/µL Promega sequencing grade modified trypsin in 50 mM ammonium bicarbonate.
8. Extraction solution: 5% formic acid in 50% acetonitrile.
9. Microfuge tubes. We use plain 1.5 ml tubes and low binding 0.65 ml. Rinse all tubes with water, ethanol, water, ethanol. It appears that thorough washing removes acetonitrile soluble material which forms a layer on aqueous solutions and interferes with evaporation.
When removing solution from gel pieces, microfuging does not seem to make any significant difference in the amount of liquid removed.
With faintly stained gel pieces, watch carefully to ensure that the gel piece stays in the tube and does not stick to a pipet tip.
When removing wash and alkylation solutions, using the same pipet tip does not seem to give cross contamination, although as a precaution, the tip is changed between different groups of samples. Use a fresh tip for each sample when removing peptides.
- Cut bands from gel as closely as possible. Divide into smaller pieces.
- Destain the bands in 500 µL Destain overnight at room temperature.
- Remove Destain and replace with 200 µL Destain for 2 to 3 h.
- Remove the Destain (discard), and dehydrate gel slices in 200 µL acetonitrile. Gel pieces should turn opaque-white within 5 minutes. If they don’t, then remove acetonitrile and add another 200 µl of acetonitrile.
- Remove acetonitrile (discard) and evaporate any residual acetonitrile in SpeedVac (2 to 3 min).
- Reduce the gel pieces in 30-50 µL 10 mM DTT for 30 min. at RT.
- Remove DTT solution.
- Alkylate in 30-50 µL 50 mM iodoacetamide for 30 min. at RT.
- Remove iodoacetamide solution.
- Wash with 100 µL 100 mM ammonium bicarb for 10 min.
- Remove wash.
- Dehydrate gel slices in 200 µL acetonitrile. Gel pieces should turn opaque-white within 5 min. If they don’t, then remove acetonitrile and add another 200 µl of acetonitrile.
- Remove acetonitrile and rehydrate by swelling in 100 µL 100 mM ammonium bicarb for 10 min.
- Remove ammonium bicarb.
- Dehydrate gel slices in 200 µL acetonitrile.
- Remove acetonitrile and add another 200 µL aliquot of acetonitrile.
- Remove acetonitrile.
- Dry gel pieces in SpeedVac (2 to 3 min).
- Prepare trypsin. 20 µg of Promega trypsin in 1000 µL ice cold 50 mM ammonium bicarb (trypsin concentration = 20 ng/µL). Keep ice cold.
- Add 30 to 50 µL of the trypsin solution to cover the gel pieces and incubate for 5 to 10 min on ice. Watch that gel pieces appear re-swollen. (The idea is to allow the trypsin to move into the gel but not begin digestion.)
- Remove any excess trypsin solution and add 5-20 µl 50 mM ammonium bicarb. React overnight at 37 °C.
- Extract with 30µl 100 mM ammonium bicarb. Vortex. Incubate 10 min., microfuge, and take off supernatant to a clean 0.5 mL microfuge tube.
- Extract the peptides by adding 30 µL extraction solution. Incubate for 10 min and collect the extract to the same microfuge tube.
- Repeat the extraction with a second aliquot of the extraction solution, combining the extracts in the microfuge tube.
- Evaporate the sample to 20 µL (do not go to complete dryness). Commonly tubes are evaporated to 20 µl after 45 min, but at times may take much longer. To MS.