2023 In gel digest with TCEP / CAA

In-gel Digest

Adapted from Goodman et al. “Updates of the In-Gel Digestion Method for Protein Analysis by Mass Spectrometry” 2018 paper.

Transfer cut gel cubes to a 1.5 ml tube. Use as much of each solution detailed below to cover the gel pieces. This will obviously depend on each project so use common sense. 100 µl is normally sufficient. Mixing should be performed on a Thermomixer or equivalent.

Make up fresh 100 mM ammonium bicarbonate (AMBIC) by dissolving 791 mg of AMBIC in 100 ml of water.

To 5 ml of 100 mM AMBIC, add 5 ml of ethanol (EtOH). This will create 10 ml of 50 % EtOH in 50 mM AMBIC.

De-stain

Round 1 - add 50 % EtOH in 50 mM AMBIC to each sample, then mix at 25 °C for 15 minutes at 650 rpm. Remove solution and discard.

Dehydrate gel bands by adding 100 % EtOH to each sample, then mix at 25 °C for 5 minutes at 650 rpm. Remove EtOH and discard.

Round 2 - repeat as per round 1. Two rounds tend to be sufficient but heavily stained bands may require further rounds of de-staining.

Reduction and alkylation

Make up a 100 mM solution TCEP in water. The molecular mass of TCEP is 250.187 g/mol.

Make up a 400 mM solution of chloroacetamide in water. The molecular mass of chloroacetamide is 93.51 g/mol.

To 800 µl of water, add 100 µl of 100 mM TCEP and 100 µl of 400 mM chloroacetamide. This will create 1 ml of 10 mM TCEP and 40 mM chloroacetamide in water.

Reduce and alkylate simultaneously by adding the solution of 10 mM TCEP and 40 mM chloroacetamide. Incubate at 70 °C for 5 minutes at 650 rpm. Remove TCEP/chloroacetamide solution & discard.

Washing

Wash samples by adding 50 % EtOH in 50 mM AMBIC the mix at 25 °C for 15 minutes at 650 rpm. Remove solution and discard.

Dehydrate gel bands by adding 100 % EtOH to each sample, then mix at 25 °C for 5 minutes at 650 rpm. Remove EtOH and discard.

Trypsin digestion

Example: 10 µg of protein to be digested so 1:50 ratio means 200 ng of trypsin should be added.

Add 50 µl of 4 ng/µl trypsin in either 50 mM AMBIC or 50 mM HEPES solution to each sample. Add additional 50 mM AMBIC if required to cover the gel pieces. Incubate at 37 °C for at least 3 hours at 650 rpm.

Peptide Extraction – approx. 15 minutes

Round 1 - make up a 25 % acetonitrile solution by adding 250 µl of acetonitrile to 750 µl of water.

Add 25 % acetonitrile solution to each sample and sonicate for 5 minutes in a water bath. Remove the solution and transfer to a fresh 0.65 ml tube.

Round 2 - repeat as per round 1 and combine the solution with the solution from round 1.

Round 3 - add 100 % acetonitrile to each sample and sonicate for 5 minutes in a water bath. Combine with the solution from rounds 1 and 2.

Dry the peptide solution using a vacuum centrifuge for one hour.

Desalt with µ-C18 tips.

Dry the peptide solution using a vacuum centrifuge for 30 minutes.

Re-suspend in 7 µl of solvent A ready for injection on the LC-MS instrument.