丙烯酰胺胶中肽提取和蛋白消化技术Digestion of Proteins and Extraction of Peptides from an Acrylamide Gel

Digestion of Proteins and Extraction of Peptides from an Acrylamide Gel

• Acetonitrile
• Ammonium bicarbonate (NH₄ HCO₃ ), 100 mM
• CaCl₂ , 1 M
• Digestion buffer
  • 50 mM NH₄ HCO₃ , 5 mM CaCl₂
• Formic acid 90%, 5%
• Milli-Q water
• Iodoacetamide (IAA), 55 mM (prepare fresh)
• K₃ Fe(CN)₆
• Na₂ S₂ O₃ ・ 5H₂ O
• Proteins in gel slices as obtained in Protocol 1, contained in a microfuge tube
• Tris (2-carboxyethyl)-phosphine hydrochloride (TCEP)
• Trypsin, sequencing grade, modified

• Speed vacuum
• Incubator, preset to 37°C

1. If proteins were visualized by silver stain, first remove silver stain (steps 1-3). However, if proteins were visualized by Coomassie, begin at step 4. If the silver-stained gel slice was stored in acetic acid, remove the storage solution and rinse the slice with water.
2. For each gel slice, prepare a 30-mM solution of K₃ Fe(CN)₆ and a 100-mM solution of Na₂ S₂ O₃ ・ 5H2O. Mix 200 µl of each solution together (total 400 µl) and transfer the mixture to the microfuge tube containing the gel piece. Incubate for 5 min with gentle vortexing.
3. Remove the liquid and wash each gel slice 3 times with 500 µl of water. Incubate the slices in 100 µl of 100 mM NH₄ HCO₃ for 30 min. Discard the liquid and proceed with step 4.

Removal of Coomassie Staining and Treatment of Gel Slices

1. At this point, the gel slices are either stained with Coomassie or have just had the silver stain removed. In either case, wash the gel slices with water for 15 min. All steps should be performed with light vortexing. The volume of solvents added in the wash and elution steps should be twice that of the cut gel pieces (~100 µl).
2. Remove the liquid remaining from step 4 and add 100 µl of water, and then 100 µl of acetonitrile to the gel pieces. Incubate for 15 min at room temperature.
3. Remove the liquid and add a sufficient volume of acetonitrile to cover the gel pieces (~100 µl). The pieces will dehydrate, shrinking and turning white and sticky. After this, remove the acetonitrile.
4. Rehydrate the slices by adding 100 µl of 100 mM NH₄ HCO₃ . Wait 5 min, and then add 100 µl of acetonitrile. Incubate for 15 min at room temperature. If Coomassie staining was used to visualize the proteins and a strong blue color persists, incubate for a further 15 min with continuous gentle vortexing. Remove the liquid and dry down the gel pieces in a speed vacuum for 15 min.

Reduction, Alkylation, and In-gel Digestion

1. Rehydrate the gel pieces using a sufficient volume of 10 mM TCEP in 100 mM NH₄ HCO₃ to cover them (~100 µl). Incubate for 20-30 min at room temperature.
2. Remove the liquid and quickly add the same volume (~100 µl) of 55 mM IAA in 100 mM NH₄ HCO₃ . Incubate for 30 min at room temperature in the dark. It is important that the IAA is freshly prepared.
3. Remove the liquid and dehydrate the gel by adding 100 µl of acetonitrile. After the gel pieces have shrunk and turned white and sticky, remove the acetonitrile. Add 100 µl of 100 mM NH₄ HCO₃ and incubate for 5 min at room temperature to allow rehydration.
4. Add 100 µl of acetonitrile to create a 1:1 mix of 100 mM NH₄ HCO₃ and acetonitrile, and incubate for 15 min.
5. Remove the liquid and dry the gel slices completely in a speed vacuum, for at least 30 min. Rehydrate in 100 µl of digestion buffer containing 12.5 mg/ml trypsin. Incubte at 37°C overnight.

Extraction of Peptides

1. Centrifuge the trypsin digests briefly in a microcentrifuge and transfer the peptides (in the supernatant) to a 0.5-ml microfuge tube and reserve. In the meantime, add 100 µl of 25 mM NH₄ HCO₃ to the gel pieces (make sure they are covered by the solution) and incubate for 15 min. Then add 100 µl of acetonitrile and incubate for a further 15 min.
2. Recover the liquid and transfer it to the 0.5-ml tube containing the peptides. Begin drying the peptides in a speed vacuum, while continuing with steps 14 and 15. Add 100 µl of 5% formic acid to the gel slices and incubate for 15 min. Next, add 100 µl of acetonitrile to the slices and incubate for 15 min.
3. Recover the supernatant from the slices and transfer it to the 0.5-ml tube containing the peptides. Repeat step 14. Continue drying the sample in the 0.5-ml microfuge tube in the speed vacuum for ~2 hr, until there is only 10 µl remaining. At this point, the peptides are ready for analysis. Analysis can be done by ESI MS, ESI MS/MS (tandem MS), or MALDI/TOF.