LC-MS/MS Protocol for Peptide Analysis

LC-MS/MS analysis was carried out as previously described^{20 (link),62 (link)}, with some modifications. Each obtained peptide mixture was resuspended in 0.1% TFA and injected into an analytical column (Zorbax 300SB-C18 75 um i.d. × 15 cm column; Agilent, Germany) via a trap column (Zorbax 300SB-C18 300 μm i.d. × 5 mm column; Agilent). The peptides were separated in an acetonitrile gradient of buffer A (0.1% formic acid in water) and buffer B (0.1% formic acid in pure acetonitrile) at a constant flow rate of 0.2 μl/min, using an Agilent 100 series nano HPLC system coupled on-line to a LTQ ion-trap mass spectrometer (Thermo Fisher Scientific). The gradient commenced with 5% B, rose linearly to 40% B over 100 min, increased to 80% B over 1 min, and then isocratically increased to 80% B over 15 min. A full-scan mode (m/z 350–1600) was enabled, and each survey MS scan was followed by three MS/MS scans using the 30-sec dynamic exclusion option set. The utilized ESI-Q-TOF ion source parameters were as follows: ion spray voltage, 2.2 kV; capillary voltage, 24 V; and capillary temperature, 200 °C. The rolling collision energy was set to 35%.