Reduction of Interference Ions Generated in the Enrichment Process of Biotinylated Peptides using Tamavidin 2-REV

After tryptic digestion of proteins of RAW264.7 cells expressing TurboID-STING, biotinylated peptides were captured with Tamavidin 2-REV magnetic beads and eluted competitively with excess biotin. When the peptide samples were subjected to LC-MS/MS, high intensity peaks of interference and/or contaminant ions were observed at the MS1 level. To reduce interference ions, we compared the following conditions: surfactants to solubilize proteins (RapiGest vs. PTS); trypsin inactivation (Pefabloc SC vs. heat); beads pre-wash (none vs. 10% ACN); and biotin clean-up (none vs. GL-Tip SDB). To confirm that some contaminant ions were derived from the biotin solution, the biotin solution or biotin-free solution was desalted using GL-Tip SDB and directly analyzed by LC-MS/MS. We assessed reproducibility of each condition by three technical replicates.