This document provides a general guideline for preparation of samples to be submitted to proteomics facility. Users are encouraged to contact the facility for concerns that are not covered to this guide line. A submission form should be filled and attached to each submission.
Using a clean razor blade, cut the protein bands of interest from the gel, eliminating as much polyacrylamide as possible. Place the gel slices into a microcentrifuge tube that has been prewashed twice with 50% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA). Note: To avoid contamination, razor blades, staining containers and all other equipment that comes into contact with the gel or gel slices should be cleaned with laboratory detergent (e.g., Alconox®) and rinsed well prior to use.
Destain the gel slices twice, with 0.2 ml of 100 mM NH4HCO3/50% ACN for 15 minutes at 37°C.
Dehydrate the gel slices for 2 minutes at room temperature in 100 μl 100% ACN. At this point, the gel slices will be much smaller than their original size and will be white or opaque in appearance.
If protein has denatured and alkylated prior to electrophoresis, move to step 7. Otherwise, add 100 μl 50 mM DTT (dithiothreitol) in 50 mM ammonium bicarbonate, incubate at 65 °C for 1 hr. Remove liquid, dehydrate gel as in step 3.
Alkylate with 100 μl 55 mM iodoacetamide in 50 mM ammomium bicarbonate, incubate at room temperature in the dark for 1 hr. Remove liquid, dehydrate gel as in step 3.
Resuspend the Trypsin at 1μg/μl in 50mM acetic acid, then dilute in 50mM NH4HCO3/10% ACN to 20μg/ml. Preincubate the gel slices in a minimal volume (10–20μl) of the trypsin solution at room temperature or on ice (do not exceed 30°C) for 1 hour. The slices will rehydrate during this time. If the gel slices appear white or opaque after one hour, add an additional 10–20μl of trypsin and incubate for another hour. Note: Trypsin specific activities can vary widely, depending on the manufacturer. Procedures describing the use of trypsin by weight may need to be optimized depending on the specific activity of the trypsin being used.
When gel is fully hydrated, remove excess liquid. Add enough digestion buffer (40mM NH4HCO3/10% ACN) to completely cover the gel slices. Cap the tubes tightly to avoid evaporation. Incubate overnight at 37°C.
Incubate the gel slice digests with 150μl of NANOpure® water for 10 minutes, with frequent vortex mixing. Remove and save the liquid in a new microcentrifuge tube (see Note at the end of this section).
Extract the gel slice digests twice, with 100μl of 50% ACN/5% TFA (with mixing) for 60 minutes each time, at room temperature (see Note at the end of the section).
Pool all extracts (Steps 10 and 11) and dry in a Speed Vac® at room temperature for 2–4 hours (do not exceed 30°C).
Dissolve the target protein in 6M guanidine HCl (or 6–8M urea or 0.1% SDS), 50mM Tris-HCl (pH 8), 2–5mM DTT (or β-mercaptoethanol).
Heat at 95°C for 15–20 minutes or at 60°C for 45–60 minutes. Allow the reaction to cool.
For denatured proteins, add 50mM NH4HCO3 (pH 7.8) or 50mM Tris-HCl, 1mM CaCl2 (pH 7.6), until the guanidine HCl or urea concentration is less than 1M. If SDS is used, dilution is not necessary. For digestion of native proteins, dissolve the protein in buffer with a pH between 7 and 9.
Add Trypsin Gold to a final protease:protein ratio of 1:100 to 1:20 (w/w). Incubate at 37°C for at least 1 hour. Remove an aliquot and chill the remainder of the reaction on ice or freeze at –20°C.
Terminate the protease activity in the aliquot from Step 4 by adding an inhibitor. Alternatively, precipitate the aliquot by adding TCA to a 10% final concentration. The reaction can also be terminated by freezing at –20°C. Trypsin can also be inactivated by lowering the pH of the reaction below pH 4. Trypsin will regain activity when the pH is raised above pH 4 (4).
Determine the extent of digestion by subjecting the aliquot in Steps 4 and 5 to reverse phase HPLC or SDS-PAGE.
If no inhibitors were added to the remainder of the reaction and further proteolysis is required, incubate at 37°C until the desired digestion is obtained (9). Reducing the temperature will decrease the digestion rate. Incubations of up to 24 hours may be required, depending on the nature of the protein. With long incubations, take precautions to avoid bacterial contamination.