Phosphopeptide Enrichment: An Antibody-based Immunoprecipitation Technique to Separate Specific Phosphorylated Peptides from Complex Peptide Mixtures
Transcription
Phosphorylation of specific amino acid residues, like tyrosine, generates phosphopeptides that play critical roles in regulating several cellular signaling pathways. Studying them allows the identification of potential biomarkers for cancer therapy.
To isolate phosphopeptides, begin with a lyophilized powder of purified peptide digests extracted from cancer tissue. Resuspend the powder in a suitable buffer to prepare a uniform peptide suspension. Maintain a neutral pH to ensure peptide stability.
Prepare a slurry consisting of hydrogel beads conjugated to specific anti-phosphopeptide antibodies in the desired buffer. Add the bead slurry to the peptide suspension. The antibodies immobilized on the beads recognize and bind to their specific phosphorylated amino acid residues within the phosphopeptides, forming complexes.
Centrifuge the mixture to collect the phosphopeptide-bead complexes in the pellet. Remove the supernatant containing unbound peptides or non-specific phosphopeptides. Add a suitable low pH elution buffer to the complexes and incubate. The low pH weakens the interaction between the phosphopeptide residues and antibody-bead complexes, releasing the peptides into the solution.
Transfer the mixture to a pre-assembled filter cartridge. Centrifuge to elute the released phosphopeptides into the collection tube. Vacuum concentrate the sample at an elevated temperature to remove the buffer and obtain a concentrated dry pellet of pure phosphopeptides.
