Easy spray nano

The proteomic pipeline, including protein reduction, alkylation, digestion, desalting, and nano LC-MS/MS analysis, was performed as previously reported (Hernández-Pérez et al., 2019 (link)), with minor modifications. Comparative proteomics was carried out with Isobaric tags for relative and absolute quantitation (iTRAQ) and synchronous precursor selection-MS³ in an UltiMate 3000 RSLC system (Dionex©) coupled to an Orbitrap Fusion Tribrid (Thermo-Fisher Scientific©) mass spectrometer equipped with an “EASY Spray” nano ion source (Thermo-Fisher Scientific©). iTRAQ 8-plex reagents (cat. number 4381663, Sigma-Aldrich©) with varying molecular weights were applied as isobaric labels for the comparative quantification of proteins between the digested samples: 113, 114, 115, and 116 for the healthy group and 117, 118, 119, and 120 for the infected group, according to the manufacturer’s protocol.

Analysis of peptide mixtures was carried out using an Acclaim^TM PepMap^TM RSLC column with an inner diameter of 50 cm × 75 μm (Thermo Fisher Scientific) using a 2-hour acetonitrile gradient in 0.1% v/v formic acid in dH₂O, flow rate of 250 nl/min and an Easy-nLC 1000 coupled to a Q Exactive Hybrid Quadrupole-Orbitrap^TM Mass Spectrometer via an EASY-Spray nano-ESI source (all Thermo Fisher Scientific). The Q Exactive Hybrid Quadrupole-Orbitrap^TM Mass Spectrometer was run in data-dependent mode with survey scans acquired at a resolution of 75,000 at m/z 200. Up to 10 of the highest abundance isotope patterns with a charge of +2 or higher were selected to proceed from the survey scan (isolation window of m/z 3.0) to fragmentation by higher-energy collision dissociation with normalized collision energies of 25. Maximum ion injection times for the survey scan and the MS/MS scan (resolution of 17,500 at m/z 200) were 20 and 120 ms, respectively. Ion target value for MS was 10⁶ and for MS/MS was 10⁵. The intensity threshold was 8.3 × 10².