Sequential Digestion with Different Proteases

We examined three combinations of different proteases for digestion of proteins from TurboID-STING-expressing cells: trypsin alone, Lys-C followed by trypsin, and Glu-C followed by trypsin. First, we tested whether PTS buffer used to solubilize precipitated proteins is compatible with digestion with these proteases. Next, biotinylated peptides were enriched after digestion with the three combinations of different proteases. We assessed reproducibility of each combination using three technical replicates.