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Materials
NOTE: To avoid or reduce contamination with human keratins, cleanliness is very important throughout the procedure. Be very careful to avoid dust particles, always wear gloves and preferably work in a laminar-flow hood.
Excision and washing
1. Excise protein bands/spots of interest from the stained polyacrylamide gel. Use a clean scalpel, and cut out the darkest part of the band, leaving the light edges behind. Cut each gel slice into small pieces (~1 mm3) using a scalpel or cleaned razor blade, and put into a 0.5 mL tube. Also cut out and dice a gel piece from a protein-free region of the gel, for a parallel control digestion.
If you are not using conventional Coomassie stain, we recommend Invitrogen Colloidal Blue (P/N LC6025) (Coomassie) stain. We do not recommend silver-stained gels at present. The sensitivity of Colloidal Blue is roughly 1/3 to 1/2 of that of silver stain. If you use conventional Coomassie, stain and destain the gel with <10% acetic acid. Higher concentrations will interfere with digestion.
The efficiency of protein digestion and peptide extraction depends on properties of the specific protein (degree of hydrophobicity, etc.). In general, picomole levels are preferred to lower levels, and more is almost always better; however, more gel pieces is not necessarily better. Overall efficiency is much higher with a single concentrated gel band than with several more dilute bands.
At this point, the gel slices shrink and become white. This visual criterion should be used to determine whether or not additional washes should be performed.
3. Dry gel pieces for ~30 min in a vacuum centrifuge.
(optional) Reduction and alkylation
We do this if the protein may contain disulfides; this procedure inevitably results in some loss of sample and is often not necessary. We avoid it if possible.
4. Add enough 10 mM DTT solution to cover the gel pieces, and reduce for 1 hr at 56°C.
5. Cool to room temperature and replace the DTT solution with roughly the same volume of 55 mM iodoacetamide solution. Incubate for 45 min at room temperature in the dark with occasional vortexing.
6. Wash gel pieces (rehydrate) with ~100 μL of 25 mM ammonium bicarbonate, pH 8, for 10 min while vortexing, and dehydrate with ~100 μL of 25 mM ammonium bicarbonate/50% acetonitrile. Repeat rehydration and dehydration.
7. Remove the liquid phase and dry the gel pieces in a vacuum centrifuge for 20 min.
Digestion
8. Rehydrate gel pieces in one volume of 0.05 to 0.1 mg/mL (2-4 μM) trypsin solution. Do not add more solution than can be absorbed by the gel pieces.
The volume needed can be estimated by calculating the total gel volume excised (e.g., 2 mm x 8 mm x 1 mm = 16 mm3 = 16 μL).
The enzyme-to-substrate ratio employed for in-gel digestions (>1:10) is greater than for in-solution digestions due to hindered enzyme access to the protein substrate in the gel. The relatively low buffer concentration (25 mM) is used to reduce the possibility of subsequent salt interference with ionization in the mass spectrometer.
9. If necessary, overlay the rehydrated gel pieces with a minimum amount of 25 mM ammonium bicarbonate to keep them immersed throughout digestion.
10. Incubate 6 to 8 hr at 37°C.
Peptide recovery
11. Add a volume of water equal to twice the volume of the gel piece, vortex for 10 min. Use a gel-loading tip to remove the peptide solution and transfer it to a 0.5 mL tube.
12. Perform two additional extractions using the same volume of 5% TFA/50% acetonitrile.
Formic acid (5%) may be used as an alternative to TFA. Some labs prefer to use silanized tubes.
13. Concentrate recovered peptides by reducing the final volume of the extracts to ~5 μL in a vacuum centrifuge.
Note that evaporating the solution to complete dryness will result in some loss of peptides. The recovered peptides are now ready for desalting (Zip-Tips). They can be stored at -20°C until the next step can be performed.