In Solution Digestion

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.

Peptide 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.