Peptide Purification: An RP-HPLC-based Technique to Extract Peptides from Digested Protein Lysates

1. Reverse Phase Extraction

1. Start with a pre-digested protein lysate in a glass vial. Filter the sample by using a 15 mL 10 kDa cutoff filter. Centrifuge the sample at 3,500 x g using the swing bucket rotor (or 3,500 x g in a fixed angle rotor) at 15 °C until the retentate volume is less than 250 µL (this takes approximately 45 – 60 min). Collect the flow-through and discard the retentate.
   NOTE: The experiment can be paused here. Freeze the samples at -80 °C for later use.
2. To acidify the sample, add approximately 20 µL of 5% trifluoroacetic acid (TFA) per mL of lysate. Mix them well and measure the sample pH by using pH strips. Adjust pH to 2.5 using 5% TFA.
3. Connect the shorter end of a C-18 column to a vacuum manifold. Set the vacuum between 17 and 34 kPa (or according to the manufacturer’s instructions). Using glass pipettes, wet the column with 3 mL of 100% acetonitrile (ACN). Do not let the column dry.
4. Using glass pipettes, equilibrate the column with 6 mL of 0.1% TFA applied as 2x 3 mL. Load the acidified sample into the column. Do not add more than 3 mL at a time. Adjust the vacuum to target about 1 to 2 drops per second.
5. Using glass pipettes, wash the column with 9 mL of 0.1% TFA applied as 3x 3 mL. Elute the column with 2 mL of 40% ACN, 0.1% TFA. Collect two 2 mL fractions into glass culture tubes. Discard the column.
6. Cover the eluate tubes with parafilm and punch 3-5 holes on the cover using a 20G needle. Freeze the eluate on dry ice for at least 30 min until it completely solidifies.
7. Lyophilize the fractions overnight. On the following day, make sure the samples are completely dry before stopping the lyophilizer. Store the tubes in a 50 mL conical tube with delicate wipes at -80 °C.