Mass spectrometry analysis

Each peptide mixture was twice subjected to reversed-phase liquid chromatography followed by tandem mass spectrometry (LC–MS/MS) analysis with an UltiMate 3000 nanoHPLC (Thermo Scientific®) coupled online with an Exploris 240 Orbitrap mass spectrometer (Thermo Scientific®). The peptide mixture was chromatographically separated on a column (15 cm in length with a 75 μm I.D., C18-AQ 3.0 μm resin, SNC20442712 - Thermo Scientific) with a flow of 250 nL/min from 1% to 40% ACN (acetonitrile) in 0.1% formic acid, in a 120 min gradient. The Exploris 240 Orbitrap was set to the data-dependent acquisition (DDA) mode to automatically switch between full scan MS and MS/MS acquisition with 30 s dynamic exclusion. Survey scans (200–2000 m/z) were acquired in the Orbitrap system with a resolution of 60,000 at m/z 200. The most intense ions captured in a 2 s cycle time were selected, excluding those unassigned and in a 1 + charge state, sequentially isolated and HCD (Higher-energy collisional dissociation) fragmented using a stepped normalized collision energy of 25, 30, and 35. The fragment ions were analyzed with a resolution of 15,000 at 200 m/z. The general mass spectrometric conditions were as follows: 2.5 kV spray voltage, no sheath or auxiliary gas flow, heated capillary temperature of 40 °C, predictive automatic gain control (AGC) enabled, and an S-lens RF level of 40%. Mass spectrometer scan functions and nLC solvent gradients were controlled by the Xcalibur 4.1 data system (Thermo Scientific®).