Proteomic Analysis of Digested Samples

As previously described, all digested samples were analyzed by nLC-MS/MS [16 (link)]. Briefly, peptides were chromatographically separated using the EASY-nLC II system (Thermo Fisher Scientific, Waltham, MA, USA) using A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile as the mobile phase. Samples were chromatographically resolved using a 5–70–95% B gradient for 80 min at a flow rate of 300 nL/min. Peptides were analyzed using LTQ Orbitrap XL (Thermo Fisher Scientific Inc., Bremen, Germany) in a data-dependent mode with the 10 most intense precursors subjected to fragmentation by collision-induced dissociation.