Easy nanolc system

Each HILIC fraction was resuspended in 0.1% FA, and peptides were loaded on an in-house packed pre-column (2 cm × 100 μm inner diameter 5 μm) using an Easy-nanoLC system (Thermo Fisher Scientific) and separated by gradient from 3 to 28% solvent B in 100 min, 28–45% in 20 min, 45–100% B in 5 min and 8 min at 100% B. (A = 0.1% FA; B = 90% ACN, 0.1% FA) at a flow of 250 nL/min on analytical Reprosil-Pur C18-AQ column (17 cm × 100 μm 3 μm inner diameter). The Easy-nanoLC system was connected online to a QExactive HF Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) operating in positive ion mode and using data-dependent acquisition. The Orbitrap acquired the full MS scan with an AGC target value of 3 × 10⁶ ions and a maximum fill time of 100 ms. Each MS scan was acquired at 120,000 resolution with a mass range of 400–1600 Da. The 10 most abundant peptide ions were selected for HCD fragmentation (collision energy: 29). Fragmentation was performed at 60,000 resolution for a target of 1 × 10⁵ and a maximum injection time of 200 ms using an isolation window of 1.2 m/z, dynamic exclusion of 30 s and fixed first mass 110 m/z.

Enzymatic digestion. The monomeric protein samples were prepared by diluting 20 μl of protein (0.5 μg μl⁻¹) in 20 μl of either PBS pH 7.4, PBS pH12.8 or PBS pH12.8+30 mM dimedone. The high-pH samples had a calculated final pH=12.5. In-solution digestion with trypsin of 5 μg of total protein monomers was performed before subsequent analysis by MS. Trypsin digestion at a ratio of 1:20 (enzyme:substrate) was carried out for 2 h at 37 °C.
LC-MS/MS. Chromatographic separations were performed using an EASY NanoLC system (ThermoFisherScientific, UK). Peptides from a total protein amount of 2 μg on column were resolved by reversed phase chromatography on a 75 μm C18 column using a three step linear gradient of acetonitrile in 0.1% formic acid. The gradient was delivered to elute the peptides at a flow rate of 300 nl min⁻¹ over 60 min. The eluate was ionized by electrospray ionization using an Orbitrap Velos Pro (ThermoFisherScientific, UK) operating under Xcalibur v2.2. The instrument was programmed to acquire in automated data-dependent switching mode, selecting precursor ions based on their intensity for sequencing by collision-induced fragmentation using a Top20 CID method. The MS/MS analyses were conducted using collision energy profiles that were chosen based on the mass-to-charge ratio (m/z) and the charge state of the peptide.

Gel sections were digested with bovine trypsin (Sigma) following reduction and alkylation of cysteine bonds with dithiothreitol and iodoacetamide (Sigma). Peptide extracts were subjected to chromatographic separation by C18 reversed-phase nano-trapping (Acclaim PepMap100 C18 Trap, 5 mm × 300 µm) and nano-analytical columns (EASY-Spray PepMap C18, 2 μm 100 Å, 75 µm × 15 cm) on an EASY NanoLC system (ThermoFisher) using a three-step linear gradient at a flowrate of 250 nl/min over 60 min. The eluate was ionized by electrospray ionization using an Orbitrap Velos Pro (ThermoFisher) operating under Xcalibur v2.2. Precursor ions were selected according to their intensity using the collision-induced fragmentation employing a Top20 CID method. Raw spectral data was processed using Proteome Discoverer (v1.4) against the Uniprot ‘All Taxonomy’ database under the Mascot 2.2 algorithm (Matrix Science). Results were analysed using Scaffold Software (version 4.11.0; Proteome Software).
This study was carried out in compliance with relevant institutional, national, and international guidelines, and legislation.