Procedure for In-Solution Digestion

Before proceeding, review Contamination Issues

Solution recipes you will need are here.

Necessary resources for the digestion protocol are here.

⦁ Transfer 50 ug of total protein to a new microcentrifuge tube.
⦁ Normalize the sample concentration with 50 mM AmBic to reach 50 µL (1 μg/μL of final concentration).
⦁ Add 30 µL of 0.2% solution of RapiGest SF and vortex.
⦁ Heat at 40°C while shaking for 30 minutes. Spin down condensate.
⦁ Add 10 µL of 45 mM DTT to each solution to make the final DTT concentration 5 mM.
⦁ Heat at 40°C while shaking for 30 minutes.
⦁ Remove from heat and cool for 5 minutes (to room temp). Spin down condensate.
⦁ Add 10 µL of 150 mM IAA to each solution to make the final DTT concentration 15 mM.
⦁ Incubate the solutions in the dark at room temperature for 30 minutes.
⦁ If necessary, dilute the lysate with 50 mM AmBic until the urea concentration is ≤ 1.5 M.
⦁ Add 40 µL of Trypsin/Lys-C working solution and incubate the mixture for ~16 h at 37°C (1:50 enzyme:protein).
⦁ Following digestion, centrifuge condensate to bottom of vial.
⦁ Add 5% TFA and incubate the samples for 90 min at 37°C.
⦁ Centrifuge at 14,000 g for 30 min at 6°C.
⦁ Carefully collect the supernatant and transfer to a new 1.5 mL microtube.
⦁ Desalt the peptides with C18 Spin Columns.

Petide Desalting

Samples: Before starting dilute samples with sample solution at 1 µL of solution for every 3 µL of sample for samples that are in 100% water. This will give a final concentration of 0.5% TFA in 5% ACN.

Equilibrium:

⦁ Place the column into a 2 mL tube.
⦁ Pipette 200 µl of wetting solution I into the column and centrifuge for 2 min at 250x g. Repeat once more.
⦁ Discard the throughout from collecting tube
⦁ Pipette 200 µl of wetting solution II into the column and centrifuge for 2 min at 250x g. Repeat once more.
⦁ Discard the throughout from collecting tube
⦁ Pipette 200 µl of equilibration solution into the column and centrifuge for 2 min at 250x g. Repeat once more.
⦁ Discard the throughout from collecting tube

Binding and wash

⦁ Add the sample to the column and centrifuge 2 min at 250x g.
⦁ Collect the flow through and reload into the column once more
⦁ Centrifuge 2 min at 250x g.
⦁ Pipette 200 µl of washing solution and centrifuge for 2 min at 250x g. Repeat wash step twice.

Elution:

⦁ Place column in a new 2 mL centrifuge tube.
⦁ Add 40 µL of elution solution into column and centrifuge 2 min at 250x g.
⦁ Add more 40 µL of elution solution into column and centrifuge 2 min at 250x g.
⦁ Place samples into Speed Vac and dry to near dryness (not less than 10uL). Do not dry completely!
⦁ Samples can be stored at -20°C for a few months.