LC-MS/MS Analysis of Peptides

LC–MS/MS was performed in a Dionex UltiMateTM 3000 RSLCnano coupled to a LTQ Orbitrap XL mass spectrometer via a nanospray ion source (Thermo Fisher Scientific, Auckland, New Zealand). Peptides were fractioned on a PepMap C18 column (3 μm, 300 Å, 75 μm × 15 cm; ThermoFisher Scientific, Auckland, New Zealand) on a 350 min gradient from 0 to 80% ACN in 0.1% FA at a constant flow rate of 300 nL/min and eluted into the Orbitrap via a PicoTip emitter (360 × 20 μm; New Objective, Littleton, MA, USA) at a voltage set to 1.8 kV through a transfer tube of 25 μm inner diameter [31 (link),32 (link),33 (link)]. The six most intense peptide ions from the MS scan were selected and fragmented using collision-induced dissociation (normalised collision energy, 35%; activation Q, 0.250; and activation time, 30 ms) for MS/MS scans. Dynamic exclusion was used with the following settings: repeat count, 2; repeat duration, 30 s; exclusion list size, 500; exclusion duration, 90 s [32 (link)]. The spectra was acquired using Xcalibur (version 2.1.0 SP1, Thermo Fisher Scientific, Auckland, New Zealand).