Xcalibur

For further confirming the metabolite of F2 fraction and elucidating its molecular structure, the ESI-MS/MS (LTQ XL, Thermo Electron Corporation, Waltham, MA, USA) analysis was carried out by following the protocol described earlier [36 (link)] with a few minor modifications. Briefly, 2 mg of fraction F2 was dissolved in 1 mL of methanol: acetonitrile [80:20, v/v] mixture and run, using direct injection mode at 9 μL/min. The capillary temperature was set at 288 °C. The mass range was selected at m/z 100 to 1000 in positive ionization mode for data acquisition. The collision induced dissociation energy (CID) was manually selected in the range of 5 to 30 eV for obtaining favorable fragmentation. The sheath and auxiliary N₂ gases were also adjusted manually. For the data analysis and structural elucidation, Xcalibur™ (version 3.0, Thermo Fisher Scientific, Waltham, MA, USA) and ChemDraw (version Chem Draw Ultra 8.0, PerkinElmer, Waltham, MA, USA) software were used.

LC–MS/MS analyses were performed using a UHPLC system (Vanquish, Thermo Fisher Scientific) with the UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled with the Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo). The mobile phase consisted of 25 mmol/L ammonium acetate and 25 ammonia hydroxide in water (pH = 9.75) (A) and acetonitrile (B). The auto-sampler temperature was 4 °C, and the injection volume was 2 µL. The QE HFX mass spectrometer was used for its ability to acquire MS/MS spectra on information-dependent acquisition mode in the control of an acquisition software (Xcalibur, Thermo). In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The ESI source conditions were set as follows: sheath gas flow rate, 30 Arb; Aux gas flow rate, 25 Arb; capillary temperature, 350 °C; full MS resolution, 60,000; MS/MS resolution, 7500; collision energy, 10/30/60 in NCE mode; and spray voltage, 3.6 kV (positive) or − 3.2 kV (negative).

Spectra were collected on a modified QExactive hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific) optimized for transmission of high-mass complexes (68 (link)). Protein concentration was 15 μm by monomer. Capillary voltage was 1.4 kV in positive ion mode with source temperature 200 °C and S-lens RF 200%. UHV pressure (argon) was between 1.4 × 10⁻⁹ and 1.7 × 10⁻⁹ mbar. In-source trapping fragmentation voltage ranged from −150 to −180 V. Ion transfer optics were as follows: injection flatapole 10 V, inter-flatapole lens 8 V, bent flatapole 6 V, transfer multipole 4 V, C-trap entrance lens 3 V. Nitrogen was used in the HCD cell and HCD energy was 0 V for intact spectra and tuned for optimal dissociation of each protein for CID spectra, ranging from 200 to 230 V. Resolution was kept at 17,500 at m/z = 200 for a transient time of 64 ms and the noise threshold was set to 3. For CID spectra, groupings of 30 microscans were combined to improve signal quality. Data were visualized using Xcalibur (ThermoFisher Scientific) and calibrated manually according to expected peak positions for WT αB-crystallin. Calibrated CID data were processed using UniDec software, which allowed for stoichiometric assignment and post-hoc correction for dissociated subunits (62 (link)).

In both positive and negative-ion modes, all oropharyngeal samples were subjected to metabolite separation using a UHPLC system (Vanquish, Thermo Fisher Scientific). The target compounds were separated using a Waters ACQUITY UPLC BEH Amide (2.1 mm × 100 mm, 1.7 µm) liquid chromatography column. Mobile phase A was an aqueous phase with a pH of 9.75. It contained 25 mmol/L of ammonium acetate and 25 mmol/L of ammonia hydroxide, whereas mobile phase B comprised acetonitrile. The temperature of the sample tray was 4°C, and the injection volume was 2μL.
The organic phase was injected into the column at 30°C. The elution gradients were set to 95% B, 0–0.5 min; 95–65% B, 0.5–7.0 min; 65–40% B, 7.0–8.0 min; 40% B, 8.0–9.0 min; 40–95% B, 9.0–9.1 min; and 95% B, 9.1–12.0 min. The data was gathered using Xcalibur (Thermo Fisher Scientific) on the Orbitrap Exploris 120 mass spectrometer, which can collect primary and secondary mass spectrometry data in the information-dependent acquisition mode. Other conditions for the electrospray ionization source were established as follows: capillary temperature, 320°C; collision energy, 10/30/60 in NCE mode; MS/MS resolution, 15,000; full MS resolution, 60,000; auxiliary gas flow rate, 15 Arb; and sheath gas flow rate, 50 Arb. The spray voltage was set to 3.8 and -3.4 kV for the positive-ion and negative-ion modes, respectively.

The samples were separated from proteolytic fragments by liquid chromatography (C18 column, 5 μm; pore size 100 Å; Phe-nomenex, USA) and analyzed by mass spectrometry (LCMS). The hydrolysate was fractionated on an Acclaim PepMap RSLC (50 μm × 15 cm) C18 column (2 μm; pore size, 100 Å; Thermo Scientific, USA) equipped with an Acclaim PepMap 100 (75 μm × 2 cm) C18 concentrating precolumn (3 μm; pore size, 100 Å) using an Easy Flow Nanotube nLC 1000 system (Thermo Scientific). The optained peptides were analyzed using mass spectrometer with the orbital trap (Orbitrap Elite; Thermo Scientific, Germany). Panoramic spectra were recorded in the m/z range of 300 to 2000 with a resolution of 240,000. An HCD camera was used to perform ion fragmentation. A resolution of 60,000 was adopted to record the fragmentation spectra. The data were processed using the Xcalibur (Thermo Scientific) and PEAKS Studio 7.5 (Bioinformatics Solution Inc., Canada) programs. In accordance with the search parameters for the identification of the peptide, PEAKS Studio 7.5 software was used [32 (link)].