In-gel digestion for protein identification with LC-MS (maXis impact and
amaZon)
1. Cut out the band or spot of your interest from gel as closely as possible and chop gel
pieces into 1 mm3. Transfer it to a clean microfuge tube. Do not forget to also cut a piece
of blank gel, to analyze it as your background.
2. Prepare fresh:
100 mM NH4HCO3 (237 mg in 30 mL water)
10 mM DTT (15.4 mg in 10 mL of 100 mM NH4HCO3)
55 mM iodoacetamide (101.75 mg in 10 mL of 100 mM NH4HCO3)
3. Add 200 l destaining solution (25 mM NH4HCO3, 50% acetonitrile).
4. Incubate at room temperature for 30 minutes. Discard solution and repeat 3 and 4
until the gel becomes white.
5. Remove destaining solution and dry completely in a speed-vacuum for 15 minutes.
6. Add 50 l of freshly prepared 10 mM DTT.
7. Incubate at 56ºC for 30 minutes.
8. Discard DTT solution.
9. Add 50 l 100% acetonitrile for 5 minutes. (Repeat twice.)
10. Dry in a speed vacuum for 15 minutes.
11. Add 50 l of 55 mM iodoacetamide to alkylate cysteines.
12. Incubate in the dark for 20 minutes at room temperature.
13. Centrifuge at 10,000 rpm for 2 minutes.
14. Discard the solution.
15. Wash the gel with 200 l of 100 mM NH4HCO3, vortex for 10 minutes.
16. Remove washing solution.
17. Dehydrate gel piece by adding 200 l of acetonitrile.
18. Incubate for 10 min at room temperature. Gel pieces should shrink and become
opaque-white color within 5 minutes. If they don’t, repeat this step.
19. Remove acetonitrile and dry completely in a speed-vacuum (15 minutes).
20. Add 40 l of trypsin solution: Promega sequencing grade, 20 ng/ul in 25 mM
NH4HCO3.
21. Remove excess trypsin solution and overlay the gel pieces with 40 l of 25 mM
NH4HCO3.
22. Seal the tubes using parafilm.
23. Incubate overnight (12-16 hours) at 37ºC waterbath.
NOTE: Check if the gel pieces are still overlayed by NH4HCO3 after 1 hour incubation. If
not, add more NH4HCO3 solution.
PEPTIDE EXTRACTION
24. Carefully remove parafilm from the microcentrifuge tube.
25. Spin gel pieces down at 10,000 rpm for 2 minutes.
26. Collect solution into a fresh and clean tube.
27. Add 50 l of 60% acetonitrile/ 1% trifluoroacetic acid (TFA) to the gel pieces.
28. Sonicate for 10 minutes.
29. Collect solution and combine with the solution from step 26.
30. Repeat 27, 28 and 29.
31. Dry the solution in a speed vacuum for 20 minutes.
32. Use immediately. If not, store at -20ºC.
SAMPLE DESALTING WITH ZIPTIP
33. Add 100 l of 1% formic acid (FA) into a clean tube and 10 ul elution solution (60%
acetonitrile/ 1% FA) in another clean tube.
34. Rehydrate your dry sample by adding 20-30 l of 1% formic acid (FA)/ 5%
acetonitrile.
35. Sonicate for 10 minutes.
36. Spin down sample solution.
NOTE: When using ZipTip, AVOID introducing air through the tip during the procedure,
pipetting up and down gently.
37. Set pipette to 10 l and attach a Millipore C18 ZipTip.
38. Equilibrate ZipTip: pipette up 100% acetonitrile and discard acetonitrile to waste.
Repeat this step 3 times.
39. Follow by pipetting up 1%FA and discard solution to waste. Repeat 3 times.
40. Load the digested sample by pipetting up and down 10 times.
41. Wash the ZipTip by pipetting up 1% FA and discard it to waste. Repeat it 6 times.
42. Dry in a speed-vacuum for 10 minutes.
43. Add 20-100 l (based on your initial protein quantity) of 0.1% FA/ 5% acetonitrile.
44. Sonicate for 10 minutes.
45. Spin down solution.
46. Store at -20ºC for few days if not being analyzed in the same day.