The Huck Institutes of the Life Sciences

In-solution digestion of proteins

How to digest purified proteins or protein mixtures in solution

Purified proteins or protein mixtures can be digested in solution if an additional separation step is undesirable or unnecessary.

 

Summary

Proteins in solution are denatured, their disulfide bonds are reduced, and the Cys residues are alkylated. The protein samples are incubated with trypsin for several hours, and the resulting peptides can be analyzed by MS.

Be sure to desalt or dialyze denatured, Cys-carbamidomethylated protein before adding trypsin. 

 

Materials

  • Digestion buffer: 2 mg/mL ammonium bicarbonate in water
  • Reducing reagent: 30 mg/mL TCEP, Sigma C4706 (or 15 mg/mL DTT, Sigma D0632 ) in digestion buffer
  • Alkylating reagent: 18 mg/mL IAA (Sigma I1149) prepared fresh in digestion buffer
  • Option 1: Proteomics grade trypsin (e.g. Sigma T6567-5x20UG, 5 vials x 20 ug lyophilized powder; or Promega V5113, 5 vials of frozen liquid, 40 uL of 0.5 ug/uL trypsin solution; or similar). Sigma trypsin, 20 ug can be dissolved in 20 uL of 1 mM HCl solution, aliquoted and stored at -20oC (stock solution).
    • Activated trypsin solution: dilute trypsin stock solution to 0.1 ug/uL with deionized water or digestion buffer
  • Option 2: Immobilized TPCK trypsin (e.g. Thermo 20230, 2 mL agarose gel; or similar). This product is shipped with a detailed procedure.

     

    Procedure

    Volumes are approximate. Trypsin should not exceed 5% of the total protein, provided the protein concentration range is known.

    1. Combine 15 uL digestion buffer, 1.5 uL reducing reagent, and up to 10.5 uL sample solution containing 0.025 - 10 ug protein (total volume 27 uL)
    2. Incubate in a boiling water bath for 5 min, cool to r.t., spin down to collect the sample
    3. Add 3 uL alkylating reagent and incubate in the dark at r.t. for 20 min
    • If protein sample contains detergents, salts, or chaotropic agents, perform buffer exchange after the alkylation using 3,000 MWCO centrifugal filter (Millipore UFC500396, or similar). It will be impossible to remove detergents after the digestion; and most detergents are not compatible with LC MS analysis.
    1. Add 1 uL activated trypsin and incubate at 37oC for 3 hrs. Optional: add 1 uL of fresh activated trypsin and incubate for an additional 2 hrs at 37oC or overnight.
    2. Once the incubation is complete, the peptides can be submitted for analysis or stored at -20oC.
    • Sample amount required for MS analysis is in the 100 fmol/uL range (ng/uL). Peptides at very low concentrations should not be stored for more than 2-3 days.