Home Protocols Trypsin Digestion Protocol using NEB Trypsin-ultra™ and the FASP Kit

Trypsin Digestion Protocol using NEB Trypsin-ultra™ and the FASP Kit

  1. The starting material can be cells, proteome extracts, protein complexes or pure proteins. The total amount depends on goals and complexity; for whole proteomes use between 5–50 µg, for pure proteins use between 0.5–5 µg. Please note the use of too much material can have a negative effect. Never use more than 50 µg of total protein, as filter clogging will occur.
  2. Add 200 µl of 1% SDS to each sample, vortex briefly. The use of SDS can be omitted if sample is soluble proteins or complexes.
  3. Heat at 60°C for 5 minutes.
  4. Allow samples to cool to room temperature. Add 200 µl 8 M Urea/10 mM DTT to each sample. Vortex briefly.

    5. Prepare Urea Solution: Add 1 ml 100 mM Tris HCl pH 8.5 (provided with FASP Kit) to one tube of urea (provided with FASP Kit). Vortex until all powder dissolves and add DTT to a 10 mM concentration.

  5. Rock at room temperature for 45 minutes.
  6. Transfer protein-Urea mixture to spin filter (provided in FASP Kit).
  7. Centrifuge at 14,000 x g for 15 minutes.
  8. Add 200 µl fresh Urea solution (no DTT, no SDS).

    9. Prepare Urea Solution: add 1 ml 100 mM Tris HCl pH 8.5 (provided with FASP Kit) to one tube of urea (provided with FASP Kit). Vortex until all powder dissolves.

  9. Centrifuge at 14,000 x g for 15 minutes.
  10. Discard flow-through.
  11. Add 10 µl prepared Iodoacetamide Solution and 90 µl Urea Solution (no DTT, no SDS). Incubate without mixing for 20 minutes in the dark.

    Prepare Iodoacetamide Solution: add 100 µl prepared Urea Solution (no DTT, no SDS) to 1 tube Iodoacetamide (provided with FASP Kit). Pipette up and down 10–15 times to mix well.

  12. Centrifuge at 14,000 x g for 10 minutes.
  13. Add 100 µl Urea Solution (no DTT, no SDS). Centrifuge at 14,000 x g for 15 minutes. Repeat twice.
  14. Discard flow-through.
  15. Add 200 µl 50 mM Ammonium Bicarbonate Solution (provided with FASP Kit). Centrifuge at 14,000 x g for 15 minutes.
  16. Transfer filter to new collection tube.
  17. Add 100 µl prepared Digestion Solution. Wrap tops of tubes with Parafilm to minimize evaporation. Incubate at 37°C for 4–18 hours (NO ROCKING).

    Prepare Digestion Solution: add 200 µl 50 mM Ammonium Bicarbonate Solution (provided with FASP Kit) to 20 µg Trypsin-ultra (NEB #P8101). Use 100 µl per sample for ~25–50 µg total protein. For protein quantities less than 25 µg, use 5–10 µg Trypsin-ultra and add 50 mM Ammonium Bicarbonate to bring total Digestion Solution volume to 100 µl.

  18. Add 80 µl 50 mM Ammonium Bicarbonate Solution. Centrifuge at 14,000 x g for 10 minutes.
  19. Add 50 µl 0.5 M Sodium Chloride Solution (provided with FASP kit). Centrifuge at 14,000 x g for 10 minutes.
  20. Add 170 µl 0.1% formic acid in water. Centrifuge at 14,000 x g for 10 minutes.
  21. Filtrate contains digested proteins. Total filtrate volume is 300 µl. Aliquot (150 µl x 2), freeze.
  22. Proceed with 1D or 2D-LC-MS analysis.
Notes:
  1. The FASP Protein Digestion Kit is a product of Expedeon Inc.
  2. Trypsin-ultra has been found to have very low levels of autocleavage as compared to other MS grade trypsin. Therefore, using excess Trypsin-ultra will not lower the quality of the data obtained since any excess trypsin will remain intact and be kept behind in the filter. However, it is always best to match the ratio of trypsin to protein to the best extent possible. With the FASP kit a higher trypsin concentration is suggested. Typical solution based digests normally recommend a 1:50-1:100 trypsin to protein ratio; however, when using Trypsin-ultra and the FASP Kit 1:2 to 1:10 ratios are suggested.
  3. Volumes may be adjusted based on needs and the concentration of the sample. For 1D analysis with an autosampler where minimal volume is needed it is possible to cut elution volumes in half without negative effects.