Buffers, Solutions, and ReagentsAcetonitrile Ammonium bicarbonate (NH4HCO3), 100 mM CaCl2, 1 M Digestion buffer Formic acid 90%, 5% Milli-Q water Iodoacetamide (IAA), 55 mM (prepare fresh) K3Fe(CN)6 Na2S2O3·5H2O Proteins in gel slices as obtained in Protocol 1, contained in a microfuge tube Tris (2-carboxyethyl)-phosphine hydrochloride (TCEP) Trypsin, sequencing grade, modified
Special Equipment |
Removal of Silver Stain
Before the proteins are digested, the Coomassie or silver stain must be removed from the gel.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. For each gel slice, prepare a 30-mM solution of K3Fe(CN)6 and a 100-mM solution of Na2S2O3·5H2O. 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. Remove the liquid and wash each gel slice 3 times with 500 μl of water. Incubate the slices in 100 μl of 100 mM NH4HCO3 for 30 min. Discard the liquid and proceed with step 4.
Removal of Coomassie Staining and Treatment of Gel Slices 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). 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. 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. Rehydrate the slices by adding 100 μl of 100 mM NH4HCO3. 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 Rehydrate the gel pieces using a sufficient volume of 10 mM TCEP in 100 mM NH4HCO3 to cover them (~100 μl). Incubate for 20-30 min at room temperature. Remove the liquid and quickly add the same volume (~100 μl) of 55 mM IAA in 100 mM NH4HCO3. Incubate for 30 min at room temperature in the dark. It is important that the IAA is freshly prepared. 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 NH4HCO3 and incubate for 5 min at room temperature to allow rehydration. Add 100 μl of acetonitrile to create a 1:1 mix of 100 mM NH4HCO3and acetonitrile, and incubate for 15 min. 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 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 NH4HCO3 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. 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. 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.
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