On-bead Protein Digestion for LC-MS/MS

On-bead digestion in preparation for LC-MS/MS was performed following an established protocol.[49 (link)] Digestion buffer (50 mM NH₄HCO₃) was added to the beads, and the mixture was treated with 1 mM dithiothreitol (DTT) at room temperature for 30 minutes, followed by 5 mM iodoacetimide (IAA) at room temperature for 30 minutes in the dark. Proteins were then digested overnight with 2 μg of lysyl endopeptidase (Wako) at room temperature and further digested overnight with 2 μg trypsin (Promega) at room temperature. Resulting peptides were desalted with HLB column (Waters) and were dried under vacuum.