Xcalibur software package

Untargeted liquid chromatography high resolution accurate mass spectrometry (LC-HRAM) analysis was performed on a Q Exactive Plus orbitrap mass spectrometer (Thermo Scientific, Waltham, MA) coupled to a binary pump HPLC (UltiMate 3000, Thermo Scientific). Full MS spectra were obtained at 70,000 resolution (200 m/z) with a scan range of 50–750 m/z. Full MS followed by ddMS2 scans were obtained at 35,000 resolution (MS1) and 17,500 resolution (MS2) with a 1.5 m/z isolation window and a stepped NCE (20, 40, 60). Samples were maintained at 4 °C before injection. The injection volume was 10 µL. Chromatographic separation was achieved on a Synergi Fusion 4 µm, 150 mm × 2 mm reverse phase column (Phenomenex, Torrance, CA) maintained at 30 °C using a solvent gradient method. Solvent A was water (0.1% formic acid). Solvent B was methanol (0.1% formic acid). The gradient method used was 0–5 min (10% B to 40% B), 5–7 min (40% B to 95% B), 7–9 min (95% B), 9–9.1 min (95% B to 10% B), 9.1–13 min (10% B). The flow rate was 0.4 mL/min. Sample acquisition was performed by the Xcalibur software package (Thermo Scientific). Data analysis was performed with the Compound Discoverer software package 2.1 (Thermo Scientific).

Eleven peptide fractions were analysed per group. LC-MS/MS was performed using an EASY-nLC II HPLC system (Thermo Scientific) coupled to an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific) equipped with a nano-electrospray ionisation source (Fig. 1d). Peptides were loaded onto a trap column (C18, 0.1 × 20 mm, 5 μm bead size), followed by separation on an analytical column (C18, 0.075 × 100 mm, 3 μm bead size) at a flow rate of 300 nL/min as follows: 5–15% solvent B (B) over 5 min, 15–40% B over 80 min, 40–60% B over 10 min, 60–80% B over 5 min, and 80% B over 10 min (B: 0.1% FA in HPLC-grade ACN).
Data acquisition, controlled by the Xcalibur software package (Thermo Scientific), was performed in ‘Top-20’, data-dependent, positive ion mode. Precursor ion settings included resolution at 60,000, automatic gain control (AGC) target of 1 × 10⁶, and scan rage 400–2000 m/z. Selected precursor ions were fragmented in the linear ion trap via collision-induced dissociation. Dynamic exclusion was set at 60 s to minimise repeated sequencing of the same precursors. A lock mass of 445.120025 m/z was used.

Frozen peptides were reconstituted in 3% ACN and 0.1% formic acid. A pooled sample was created by mixing 1 μL of each sample for every experiment. All samples in the same experiment were subjected together in a random order to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) for proteomic analysis as described previously⁶² (link) with the modification described below. In brief, peptides were first separated using a Thermo Scientific EASY-nLC 1200 system (Thermo Fisher Scientific) coupled to a 15 cm-long and 75 μm-diameter silica emitter as well as a ReproSil-Pur C18-AQ 120 A and 1.9 μm resin (Dr Maisch HPLC GmbH). A three-liner gradient of acetonitrile/water (containing 0.1% formic acid, at a flow rate of 300 nL/minute), first from 2% to 30% acetonitrile in 60 min, second from 30% to 97% in 10 minutes, then 97% for 10 min, was applied. The mass spectrometer was set in a data-dependent manner with an automatic switch between MS and MS/MS using the Xcalibur software package (Thermo Fisher Scientific). A mass range of 300–1500 m/z was selected for the Orbitrap analyzer.