University of Texas - College of Pharmacy

Analytical Instrumentation Facility Core

Protocol

In-gel Tryptic Digestion
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. We believe 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 Shevchenko et al. for the digestion of silver stained gels for analysis by nanospray mass spectrometry and was written up by the W.M. Keck Biomedical Mass Spectrometry Laboratory at the University of Virginia with additional modifications by our laboratory.

Reference
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).

A. Reagents (All reagents prepared fresh).

1. Destain: 50% methanol/5% acetic acid in water.
2. 100 mM ammonium bicarbonate: 100 mM ammonium bicarbonate in ultra-pure water.
3. 50 mM ammonium bicarbonate: 50 mM ammonium bicarbonate in ultra-pure water.
4. Acetonitrile.
5. 10 mM DTT: 1.5 mg/mL in 100 mM ammonium bicarbonate.
6. 50 mM iodoacetamide: 10 mg/mL in 100 mM ammonium bicarbonate.
7. Trypsin solution (on ice): 20 ng/L Promega sequencing grade modified trypsin (catalog number V511A, 20 g, porcine) in 50 mM ammonium bicarbonate.
8. Extraction solution: 5% formic acid in 50% acetonitrile.

B. Eppendorf tubes.

Siliconized 0.5 and 1.5 mL tubes. All tubes are rinsed in USP ethanol and air-dried prior to use.

C. Procedure.

Day 1

1. Cut bands from gel as closely as possible. Divide into smaller pieces.
2. Destain the bands in 0.5 mL destain overnight (or over weekend) at room temperature

Day 2

3. Remove destain and replace with 0.5 mL destain for 2 to 3 h. Skip this step if bands are no longer blue.
4. Remove the destain (discard), and dehydrate gel slices in 300 L acetonitrile. Gel pieces should turn opaque-white. If they don't, then remove acetonitrile and add another 300 L of acetonitrile.
5. Remove acetonitrile (discard) and evaporate any residual acetonitrile in SpeedVac (2 to 3 min).
6. Reduce the gel pieces in 100 L 10 mM DTT for 1 h at room temperature.
7. Microfuge and remove DTT solution.
8. Alkylate in 100 L 50 mM iodoacetamide at room temperature for 1 h.
9. Microfuge and remove iodoacetamide solution.
10. Wash with 200 L 100 mM ammonium bicarbonate for 10 min.
11. Microfuge and remove wash.
12. Dehydrate gel slices in 300 L acetonitrile approximately 5 min. Remove acetonitrile.
13. Repeat step #12.
14. Rehydrate gel pieces in 200 L 100 mM ammonium bicarbonate for 10 min.
15. Microfuge and remove ammonium bicarbonate.
16. Dehydrate gel slices in 300 L acetonitrile approximately 5 min. Remove acetonitrile.
17. Repeat step #16.
18. Dry gel pieces in SpeedVac (2 to 3 min).
19. Prepare trypsin. 20 g of Promega trypsin in 1000 L ice cold 50 mM ammonium bicarbonate (trypsin concentration=20 ng/L). Keep ice cold.
20. Add 50 to 100 L of the trypsin solution to cover the gel pieces and incubate 10 to 15 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.)
21. Add 20 L 50 mM ammonium bicarbonate. React overnight at 37 oC.

Day 3

These procedures collect the digested peptides that have diffused or are extracted into the solvent for further processing. The gel pieces may be discarded after the final extraction.

22. Centrifuge. Extract the digested peptides by removing the aqueous solution with a gel-loading pipet tip. Be careful not to suck up any gel pieces. Label tube as aqueous extraction.
23. Extract the organic fraction by adding 75 L extraction solution to the gel pieces. Incubate for 10 min, centrifuge and collect the extract in a separate 0.5 mL Eppendorf tube. Label tube as organic extraction. Again do not suck up gel pieces.
24. Repeat the extraction with a second 75 L aliquot of the extraction solution, combining the organic extracts.
25. Evaporate the samples to <25 L (do not go to complete dryness) for LC-ESI MS analysis.

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Protein Digestion on PVDF Membrane
Protein bands have to be visible by Coomassie-blue staining or even better by Ponceau-S staining:

1) Cut out bands of interest, remove completely the areas without protein.

2) If membrane is dry, wet with little methanol.

3) Destain blots completely, if possible. Destain with acetone if stained with Coomassie or with water or 0.1% acetic acid 5% ACN if stained with Ponceau-S (10-15 min). The complete removal of especially Coomassie is important since it inhibits trypsin. Then rinse with water one time.

4) Incubate blots with 200 ml of 0.25% PVP-360 in 0.1% acetic acid for 20 min.

5) Rinse the blots with copious amounts of water, 6-10 times.

6) Dry bands in open air.

7) Cut the bands into small pieces using razor blade or scissors and place them back into the same tube.

8) Add 50 ml of digestion buffer: 5ml CH3CN, 45 ml 100 mM ammonium bicarbonate, pH 8.0. Check pH at this point with small piece of pH paper.

9) Add trypsin in the ratio of 1:10, mole/mole protein, at 37oC overnight.

10) Subject sample to MALDI analysis.

Protocol developed by Melanie Lin (Perseptive Biosystems), modified by Maria Person (UT-Austin).

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Related Links
Protein digest protocols:

In-gel digest
Membrane digest
Prowl Recipes

Database Search Engines:
Prospector
Matrixscience
Prowl

Databases:

Swiss-Prot
NCBI
OWL

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Last Reviewed: August 18, 2008

Analytical Instrumentation Facility Core

Center for Research on Environmental Disease (CRED)