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Urea Solution Digest Protocol

Urea Solution Digest Protocol

Solutions Needed:

8M Urea/2M Thiourea/400mM Ammonium Bicarbonate

480mg Urea
152mg Thiourea 32mg
Ammonium Biocarbonate in 1mL water final volume

50mM Ammonium Bicarbonate

20mg Ambic in 5ml water final volume

100mM Dithiothreitol

15.4mg DTT in 1mL 50mM Ambic

100mM Iodoacetemide

18.5mg IAA in 1mL 50mM Ambic

Procedure:

  1. Dissolve protein pellet in the minimum volume necessary of 8M Urea/2M Thiourea/400mM Ambic. Volume should be no more than 50‐100 uL.
  2. Add DTT to 5mM. Pipette 5uL of the DTT solution into the protein solution if your volume is 100uL, 2.5uL if volume is 50uL.
  3. Incubate solution at 50 oC for 30min.
  4. Allow the solution to cool to room temp and add IAA to 12.5mM. Pipette 12uL of the IAA solution to the protein solution if volume is 100uL, 6uL if volume is 50uL. Incubate the solution at room temp in the dark for 20min.
  5. Quench the reaction by adding another 5uL DTT to the solution, 2.5uL if 50uL volume.
  6. Dilute solution 8x with 50mM Ambic so final concentrations are 1M Urea/250mM Thiourea/50mM Ambic.
  7. Add trypsin 1:50 (trypsin to protein). If protein concentration is not known add 100ng of trypsin solution in 50mM Ambic. Incubate overnight at 37oC.
  8. Acidify the solution to inactivate Trypsin by adding TFA to 0.5% final concentration.
  9. Desalt peptides and inject on the mass spectrometer.