This article details a method for isolating and purifying peptides from a protein lysate using reversed-phase high-performance liquid chromatography (RP-HPLC). The process involves treating the lysate with trifluoroacetic acid to enhance peptide hydrophobicity, followed by a series of washing and elution steps to obtain pure peptides.
To isolate and purify peptides, begin with a suspension of protein lysate containing a mixture of digested peptides.
Treat the mixture with trifluoroacetic acid or TFA-based reagent. This anionic or negatively charged reagent forms an ion-pair complex with basic or positively charged residues on peptides, thus masking their charge and improving peptide hydrophobicity.
Now, assemble a reversed-phase high-performance liquid chromatography, or RP-HPLC, column containing a porous silica-based stationary phase, immobilized with non-polar hydrophobic ligands. Add a peptide-compatible equilibration buffer to the column to prepare the stationary phase before sample application.
Now, load the acidified sample into the column. Within the column, the hydrophobic peptides selectively adsorb to the hydrophobic ligands of the stationary phase. Run a wash buffer through the column to elute any unbound or unwanted particles.
Next, pass a less polar organic elution buffer, with high hydrophobicity, through the column. Owing to their selective affinity towards the elution buffer, the bound peptides desorb from the column matrix.
Collect the peptide-containing eluate from the column. Lyophilize the eluate to remove the water content and obtain a dry powder of pure and stable peptides.
To acidify the sample, add approximately 20 microliters of 5% trifluoroacetic acid, or TFA, per milliliter of lysate. Mix the tube well and use a pH strip to measure the pH. If necessary, add extra 5% TFA to adjust the pH to 2.5. Next, connect to the shorter end of a C18 column to a vacuum manifold. After setting the vacuum between 17 and 34 kPa, use 3 milliliters of 100% acetonitrile and a glass pipette to wet the column.
Equilibrate the column by using a glass pipette and applying two 3-milliliter aliquots of 0.1% TFA. Then, load the acidified sample onto the column. Use three 3-milliliter volumes of 0.1% TFA to wash the column. Then, apply 2 milliliters of 40% ACN, 0.1% TFA to elute the sample and collect two 2-milliliter fractions into glass culture tubes. With parafilm, cover the eluate tubes, and use a 20 gauge needle to punch three to five holes into the cover before lyophilizing the samples overnight.
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