Reduction of Interference Ions Generated in the Enrichment Process of Biotinylated Peptides

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: (a) surfactants to solubilize proteins (RapiGest vs. PTS); (b) trypsin inactivation (Pefabloc SC vs. heat); (c) beads pre-wash (none vs. 10% ACN); and (d) biotin clean-up (none vs. GL-Tip SDB). To confirm that some contaminant ions were not derived from the digested peptides but from the biotin solution, Tamavidin 2-REV beads that had not been incubated with the digested peptides were eluted with the biotin solution or biotin-free solution, and the eluates were analyzed by LC-MS/MS.