Optimization of Elution Conditions for Biotinylated Peptides from Tamavidin 2-REV

We attempted to optimize the conditions for competitive elution of biotinylated peptides from the Tamavidin 2-REV beads. First, to optimize the temperature at which biotinylated peptides were eluted from the beads, biotinylated peptides were eluted with the biotin solution at 4, 37, 56, and 95 ºC. Second, we investigated whether the contamination of non-biotinylated peptides in the eluates could be reduced by adding a step of ‘mock’ elution with biotin-free buffer immediately before elution with the biotin solution. After the beads were incubated with a biotin-free buffer for 15 min at 37 ºC, the buffer was removed, and biotinylated peptides were eluted from the beads with the biotin solution at 37 ºC. Third, we compared biotin solutions containing various concentrations of NaCl (150, 250, 500, 1000, 2000 and 4000 mM). Finally, biotinylated peptides enriched by the original and optimized workflows from 300 µg proteins of RAW264.7 cells expressing TurboID-STING were compared by LC-MS/MS analysis.