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In-Solution Trypsin Digestion

Reagents: They should be prepared fresh right before the digestion
(Use HPLC grade solvents, highest possible grade reagents and MilliQ water for all preparations)
All procedures will be performed in the laminar-flow hood
6 M Guanidine HCl, 25 mM Ammonium Bicarbonate, pH 8.0
200 mM DTT, 25 mM Ammonium Bicarbonate, pH 8.0
200 mM Iodoacetamide, 25 mM Ammonium Bicarbonate, pH 8.0
25 mM Ammonium Bicarbonate, pH 8.0

Trypsin solution:  Use Sequencing grade modified trypsin (Promega, Cat# V5111)) . Reconstitute or dilute trypsin stock in resuspension buffer (50 mM acetic acid), keep on ice before use.

Procedure:

  1. Reconstitute the target protein (0.1-1 mg) in 10 ul of 6 M  Guanidine- HCl, 25 mM Ammonium Bicarbonate, pH 8.0 (The amount of protein depends on how complex your protein mixture is, you need to obtain a final concentration of >10 fmol/ l of each protein in the protein mixture after you finish the final step of the digestion).
  2. Add 1 ul of 200 mM DTT/ 25 mM Ammonium Bicarbonate, pH 8.0, and incubate the mixture for 1 h at room temp.
  3. Add 10 ul of 200 mM Iodoacetamide/ 25 mM Ammonium Bicarbonate, pH 8.0, gentle vortex, and incubate the mixture for 1 h at room temp in dark.
  4. Add 77.5 25 mM Ammonium Bicarbonate, pH 8.0 to reduce the guanidine HCl concentration to 0.6 M.
  5. Add Trypsin solution to a final ratio of 1:50-1:10 (w/w, trypsin:protein). Gentle vortex and incubate at 37 C for 16-20 h.
  6. Add formic acid to adjust pH to 3-4.
  7. Reduce volume to 10 ul on speed-vac.
  8. Store at -20 C.
Mass Spectrometry/Proteomics Core Laboratory

University of Kansas Medical Center
Mass Spectrometry/Proteomics Core Laboratory
1058 Hemenway
3901 Rainbow Boulevard
Kansas City, Kansas 66160