Purification of Biotinylated Peptides using SDB-SCX StageTip

We tried purification of biotinylated peptides using SDB-SCX StageTip to reduce interference ions. We compared this method with the original and optimized workflows. We assessed reproducibility of each enrichment process by three technical replicates.
Original workflow with SDB-SCX: A 15-µL slurry of MagCapture HP Tamavidin 2-REV magnetic beads per sample was washed three times with TBS2 (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl). The digested peptides containing 0.1% RapiGest SF were diluted 5-fold with TBS2 and incubated with the Tamavidin 2-REV beads in the presence of 1 mg/mL Pefabloc SC for 3 h at 4 °C. After washing five times with TBS2, biotinylated peptides were eluted with 100 μL of 1 mM biotin in TBS2 for 15 min at 95 °C twice. The combined eluates were subjected to GL-Tip SDB-SCX. After desalting with the first-stage SDB, biotinylated peptides were eluted from the second-stage SCX with 500 mM ammonium acetate and 30% ACN, evaporated in a SpeedVac concentrator, and redissolved in 0.1% TFA and 3% ACN.