In-Gel Digestion Protocol—Manual Method
This manual digestion method is essentially the same as that used in our robotic digestion using a MassPrep Digestor (MicroMass)
Starting Material: Coomassie blue, fluorescence or silver stained gel.
Procedure:
1. Gel spots (or bands) are excised with a dermal punch and placed in a 96 well V-bottom plate. Gel pieces should be small - 1.5-3 mm3. Larger gel plugs require greater volumes of trypsin buffer (but not more trypsin) and more extraction buffer volume and time for extraction; e.g. a 1.4 mm diameter gel plug from a 1.5 mm thick slab gel is 1.5 mm3. i.e. 1.5 ml. A 3 mm diameter gel plug from a 1.5 mm thick slab gel is 7 mm3. i.e. 7 ml. Faint silver spots can be pooled but no more than three per well.
2. Wash/dehydration of gel pieces: add 150 ml 50 mM ammonium bicarbonate, pH 8.5 and 50% acetonitrile (ACN) to each tube. Incubate at 37oC for 20-30 minutes. Remove and discard liquid with pipette.
3. Repeat step 2 if necessary to further destain Coomassie. (1 incubation for silver stain is usually sufficient). Gel pieces will shrink and become white when washing is complete.
3a. For silver stained plugs, prepare just before use (248 mg sodium thiosulfate in 10 ml water and 99 mg potassium ferricyanide in 10 ml water; mix 1:1 just before use; add 100 ml and incubate 20 min at 37oC.
3b. Wash gel plug 2 times with water (~10 min each), discard liquid.
4. Add 50 ml 1.5% (w/v) dithiothreitol or 5 mM tributyl phosphine in 50 mM ammonium bicarbonate, pH 8.5; incubate 20 min at 37oC.
5. Add 50 ml 5% iodoacetamide in 50 mM ammonium bicarbonate, pH 8.5; incubate 20 min at 37oC. Note: the reduction/alkylation steps (4 and 5) are done even if proteins were reduced and alkylated prior to isoelectric focusing on IPG gel strips or second dimension SDS gel electrophoresis.
6. Wash 2 times with water (~10 min each); discard liquid.
7. Dehydration: Dry gel pieces in a vacuum centrifuge (20 min) or add100 ml 50% ACN (2 times for 15 min each).
8. For dark blue spots, use 400-500 ng of Promega modified porcine trypsin. For very light spots or silver spots add 200 ng trypsin. Note: Reconstitute stock trypsin to 200 ng/ml in 50 mM acetic acid; then dilute with 50 mM ammonium bicarbonate to a working trypsin solution just before use.
9. Add enough additional 50mM ammonium bicarbonate, pH 8.5 to cover gel piece. Approximately 10-15 mL,
10. Cover plate and agitated by hand; place in a 37oC heat block; cover plate with a styrofoam insulator. Alternatively, place the 96 well plate in a zip-lok bag, seal and place in a convection type incubator. Incubate 12-16 hr at 37oC.
11. Spin sample down. To extract peptides, add 15 ml of 60% ACN/1% TFA to each gel piece. Sonicate in Branson 1200 ultrasonic waterbath for 10 minutes.
12. Centrifuge for 30 s. Transfer extracted peptides to a 96 well PCR-type plate.
13. Vacuum centrifuge extracted solution from step 12 until almost dry (IMPORTANT-do not dry for extended time ~ 20-30min)
14. Add 8 ml of 3% TFA in water to each tube. (6 mL for silver stained). Sonicate the plate in a Branson 1200 waterbath for 5 minutes.
16. Spot 0.8 ml of sample with 0.8 ml of matrix material. Matrix solution is 5 mg a-cyano and 2 mg ammonium citrate in 1 ml of 50% acetonitrile/0/1% TFA. The ammonium citrate is used to disaggregate matrix clusters greater than ca. 800 m/z.
Other Notes:
1. With low level samples, siliconized tubes (Thomas Scientific have been used successfully. In general, test for appearance of non-peptide peaks in the spectra due to plasticizers.
2. Use Optima Grade acetonitrile and water.
3. Ammonium bicarbonate solution should be made fresh each time.
4. Analyze extracted peptide samples ASAP. Losses occur with freezing of samples.
5. Sample is stable once crystallized on plate.
6. General clean environment considerations: the digest plate or microfuge tubes are opened and closed numerous time during the procedure. Make sure the work surface or test tube rack (if applicable) is clean. Work in a hood with positive air pressure if possible. Do not wear wool or silk clothing. Wash gloved hands before handling plates or tubes.
7. Calibration is done with 842.5 Da trypsin peak and 2211.1 Da trypsin peak. A parallel control can be prepared from a protein free region of the gel to identify trypsin autoproteolysis products and other non-interesting m/z peaks in the spectra.
Top of page
|
|
Protocols
The Michigan Proteome Consortium follows a standard set of protocols listed below:
Whole cell lysate
Membrane proteins
IPG 1st dimension
SDS page
Colloidal coomassie stain
Fluorescence stain
Silver stain
In-gel digestion
References and suggested reading
|
