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IN GEL TRYPSIN DIGESTION
Materials and comments:
Wash gel and eppendorf tubes with purified/distilled water. Use gloves and cover hair to avoid keratin contamination.
Volumes used should match size of excised band.
DO NOT MASH THE GEL.
Trypsin: Sequencing Grade Modified Trypsin: Promega catalog #V5111. This trypsin is in 20 µg DRIED aliquots. Do not keep any trypsin solutions, as trypsin will digest itself and lose activity.
Pipet tips: epT.I.P.S. Reloads 2-200 µl, cat no.: 022491539
epT.I.P.S. Reloads 50-5000 µl, cat no.: 022491555
Required Solutions:
These are suggested volumes for up to 25 bands. Prepare the solutions in volumes according to the number of bands in the experiment.
(A) 100 mL of 100mM NH4HCO3 =790 mg NH4HCO3 in 100 mL water (Prepare fresh.)
(B) 10 mL of 10mM DTT in 100 mM NH4HCO3 = 15.4 mg DTT in 10 mL of 100mM NH4HCO3
(C) 10 mL of 55mM iodoacetamide in 100mM NH4HCO3 = 101.6 mg in 10 mL of 100mM NH4HCO3
(D) 10 mL of 50mM NH4HCO3= 5mL NH4HCO3 of 100mM with 5mL of water
(E) 12.5 ng/µL Trypsin on ICE in 50mM NH4HCO3= 20µg Trypsin in 1.6mL of 50mM NH4HCO3 (make Trypsin solution after step 6 and keep solution on ICE). Make more depending on need or number of wells. (1.25 µg trypsin/sample)
** Try to lower to 0.5 µg trypsin/sample. 20 µg trypsin in 4 ml. 100 µL=0.05 µg trypsin.
(F) 20mL of 25mM NH4HCO3= 5mL of 100mM NH4HCO3 in 15mL of water
(G) 21mL of 5% formic acid and 50% acetonitrile = 10mL of acetonitrile in 10mL of water with 1mL of formic acid
Procedure:
1. Excise band.
2. Remove liquid from wells.
3. Dehydrate in Acetonitrile (100 µL), 30 min.
4. Remove liquid, add 200 µL of 10 mM DTT in 100 mM NH4HCO3 (B), 1 hr at 37˚C.
5. Remove liquid, add 200 µL of 55 mM Iodoacetamide in 100 NH4HCO3 (C), 45 min., RT in the dark.
6. Remove liquid, wash in 200 µL of 100 mM NH4HCO3 (A). 10 min
7. Remove liquid, dehydrate with Acetonitrile (200 µL), 30 min.
8. Remove liquid, swell in 100 mM NH4HCO3 (200 µL), 15 min.
9. Remove liquid, dehydrate with Acetonitrile (200 µL), 15 min.
10. Remove liquid, swell in 100 µL of 12.5 ng/µL Trypsin on ICE in 50 mM NH4HCO3 (E), 45 min.
11. Remove liquid and add 10 µL of 50 mM NH4HCO3 (no Trypsin) (D), 37 ˚C overnight.
In steps 12-16 peptides are washed and extracted from the gel. Label a new set of tubes and Pool all washes and extractions:
12. Wash with 100 µL of 25 mM NH4HCO3 (F).
13. Extract peptides with 100 µL 5 % formic acid, 50 % Acetonitrile (G) for 20 min.
14. Repeat step 13.
15. Extract one time with 20 µL of Acetonitrile 20min.
16. Pool post-digestion wash and all extractions and dry down (SpeedVac, no heat).
Trypsin, Promega sequencing grade modified, V5111, 100 ug, $55.
Formic acid (90 % ACS grade, Fisher) should be new and colorless. Pick up some from me if your lab stock is old.
Acetonitrile is Optima/HPLC grade from Fisher. DTT and Iodoacetamide should be new and carefully stored.
Preferred staining SYPRO Ruby stain according to standard protocol.
Destain as much as possible before or after excising band.
If you use Coomassie please excise band and destain completely.
Keratin Contamination:
Keratin contamination could be a major problem and appear as the main protein candidate in LCMSMS data. The source of contamination usually lies prior to digestion or is introduced during digestion.
Avoiding Keratin Contamination:
- Wear Gloves - Keratin is abundant in hair, nails, skin etc.
- use a Laminar flow hood if you can.
- Do not keep the tubes exposed to air for long periods of time during digestion.
- Do not handle the gel with you bare hands.
- Make fresh solvents.
- See a digestion protocol in a video (from UC Davis)
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