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

Proteins in solution are usually denatured by boiling or using denaturing buffers. During this step, the disulfide bonds must be reduced, and the sulfhydryl groups must be alkylated to prevent the disulfides from re-forming. The protein samples are then incubated with trypsin for several hours, and the resulting peptides can be analyzed by MS.

Denaturing buffers contain chaotropic agents, salts, and detergents at concentrations that inactivate trypsin. Before adding trypsin, you should desalt your protein sample and remove detergents. There are a number of the detergent removal and desalting options: detergent removal spin columns, size-exclusion and MW-cut-off spin columns, ion-exchange membranes and resins, etc.  Gel-assisted proteolysis is another option, but then it is not really an ‘in-solution’ procedure, although it does not involve electrophoresis. I thought I should mention it here in case it could be of interest to you.

You will need

Digestion buffer: 16 mg/mL ammonium bicarbonate in water

Reducing reagent: 30 mg/mL TCEP (~100 mM, Sigma C4706)  or 15 mg/mL DTT (Sigma D0632 ) in digestion buffer. NOTE: The 30 mg/mL TCEP stock solution must be prepared in 16 mg/mL (~200 mM) ammonium bicarbonate to bring up its pH. The final concentration of TCEP in the digestion mixture should be 5-10 mM.

Alkylating reagent: 18 mg/mL iodoacetamide (Sigma I1149) prepared fresh in the digestion buffer

Proteomics grade trypsin (e.g. Sigma T6567-5x20UG or Thermo Pierce 90057, 5 vials x 20 ug lyophilized powder). Trypsin, 20 ug can be dissolved in 20 uL of 1 mM HCl or 50 mM acetic acid, pH ~ 3, aliquoted and stored at -20C (stock solution).

To prepare activated (or working) trypsin solution, dilute trypsin stock solution with digestion buffer 10-fold to 0.1 ug/uL concentration.

Procedure

Volumes are approximate, it is a sample procedure after all. Trypsin should not exceed 5% of the total protein, provided the protein concentration range is known.

Combine 15 uL digestion buffer, 3 uL reducing reagent, and up to 12 uL sample solution containing 0.025 – 10 ug protein (total volume 30 uL)

Denature/reduce at 50-60 C (TCEP) or in a boiling water bath (DTT) for 5 – 10 min, cool to r.t., spin down to collect the sample

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 a 3,000 MWCO centrifugal filter. It will be impossible to remove detergents after the digestion; and most detergents are not compatible with LC MS analysis. You can find a list of MS-compatible detergents here.

Add 1-5 uL activated trypsin and incubate at 37 C for 3 hrs. Optional: add 1-5 uL of fresh activated trypsin and incubate for an additional 2 hrs at 37 C or overnight.

Once the incubation is complete, the peptides can be submitted for analysis or stored at -20 C.

Helpful tips

Minimum sample amount required for MS analysis is in the fmol/uL range (ng/uL). Solutions of peptides at very low concentrations (e.g. less than 100 fmol/uL) should not be stored for more than 1-2 days.

Always run a control along with your sample. It could be a 1 mg/mL solution of bovine serum albumin or other standard protein that you have in your lab prepared in the same buffer as your sample and taken through the entire procedure. We don’t charge for analyzing your controls.

TCEP is a great reducing reagent because it does not contain -SH groups and thus does not consume iodoacetamide during alkylation, unlike DTT. TCEP solutions in water are acidic. Depending on your buffer composition, you might observe your sample coming out of the sample tube as a soapy foam the moment you add TCEP. It is pretty much impossible to put that foam back into the tube, don’t ask me how I know. So, prepare your TCEP solutions in ammonium bicarbonate buffer to get a pH close to 8. Another note about TCEP: it should never be stored in phosphate buffers because it quickly decomposes in the presence of phosphate.

Have I missed anything? Let me know!