Exactive plus orbitrap ms

The samples, kept at 4 °C, were placed in an autosampler tray. A total of 10 μL from each sample was injected through a Synergi 2.5 micron Hydro-RP 100, 100 × 2.00 mm LC column (Phenomenex, Torrance, CA, USA) kept at 25 °C. The mass spectrometer (MS) was run in full scan mode, with negative ionization mode, using a method adapted from Lu et al. [26 (link)]. The eluent entered the MS via an electrospray ionization source attached to a Thermo Scientific Exactive Plus Orbitrap MS (Waltham, MA, USA) through a 0.1 mm internal diameter fused silica capillary tube. The samples were run with a spray voltage of 3 kV. The nitrogen sheath gas was set to a flow rate of 10 psi, with a capillary temperature of 320 °C. The AGC target was set to 3 × 10⁶. The samples were analyzed with a resolution of 140,000. A scan window of 85 to 800 m/z (mass-to-charge) was used from 0 to 9 min, and a window of 110 to 1000 m/z from 9 to 25 min. Solvent A consisted of 97:3 water:methanol, 10 mM tributylamine, and 15 mM acetic acid. Solvent B was methanol. The gradient from 0 to 5 min was 0% Solvent B, from 5 to 13 min was 20% Solvent B, from 13 to 15.5 min was 55% Solvent B, from 15.5 to 19 min was 95% Solvent B, and from 19 to 25 min was 0% Solvent B, with a flow rate of 200 µL/min.

The analyses were performed using Dionex Ultimate 3000 UHPLC coupled to an Exactive Plus-Orbitrap MS (ThermoFisher Scientific, Bremen, Germany) equipped with an HESI-II source. The chromatographic analyses of serum and tissue samples were performed using a Kinetex PFP 100 A (50 mm × 2.1 mm, 2.6 mm) and Security Guard Cartridge PFP 4 mm × 2.0 mm (Phenomenex) with a flow rate of 400 ml/min, gradient elution with 10 mM ammonium formate in 0.1% of formic acid as the mobile phase B. Gradient 0 min 5%, 4 min 45% B, and 5–6 min hold at 95%. The MS conditions were: full MS in a scan range of 50–500 m/z with positive electrospray ionization, resolution of 70,000 FWHM (scan speed 3 Hz), spray voltage of 3 kV, and ion transfer capillary temperature of 320°C.

An established untargeted metabolomics method utilizing ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) (Thermo Scientific, San Jose, CA, USA) was used to analyze water-soluble metabolites (83 (link)). A Synergi 2.6-μm Hydro RP column, 100 Å, 100 mm by 2.1 mm (Phenomenex, Torrance, CA), and an UltiMate 3000 pump (Thermo Fisher) were used to carry out the chromatographic separations prior to full scan mass analysis by an Exactive Plus Orbitrap MS (Thermo Fisher). HPLC-grade solvents (Fisher Scientific, Hampton, NH, USA) were used. Chromatographic peak areas for each detected metabolite were integrated using an open-source software package, Metabolomic Analysis and Visualization Engine (MAVEN) (83 (link), 84 (link)). Area under the curve (AUC) was used for further analyses.

The analyses were performed using Dionex Ultimate 3000 UHPLC coupled to an Exactive Plus-Orbitrap MS (ThermoFisher Scientific, Bremen, Germany) equipped with a HESI-II source. The chromatographic analyses of the serum and tissue samples were performed using a Kinetex PFP 100 A (50 × 2.1 mm, 2.6 mm) and Security Guard Cartridge PFP 4 × 2.0 mm (Phenomenex) with a flow rate of 400 ml/min, and gradient elution with 10 mM ammonium formate in 0.1% of formic acid as the mobile phase B. Gradient 0 min 5%, 4 min 45% B, 5–6 min held at 95%. The MS conditions were as follows: full MS in scan range of 50–500 m/z with positive electrospray ionization, resolution of 70000 FWHM (full width at half-maximum, scan speed 3 Hz), spray voltage of 3 kV, and an ion transfer capillary temperature of 320°C.

Peptide mapping with liquid chromatography–mass spectrometry (LC–MS) was used to profile the site-specific glycosylation sites on two NAs (A/Tanzania/205/2010 and A/Hong Kong/2671/2019). Glycopeptides containing only one specific glycan were achieved by selectively digesting with trypsin, Glu-C, Lys-C or Asp-N protease, depending on the sequence context. Of each digest product (peptide with a single glycan), 25 µg was analysed by LC–MS (Agilent AdvanceBio peptide mapping column and Thermo Q Exactive Plus Orbitrap MS). Peptide mapping data were analysed on Biopharma Finder 3.2 data analysis software. Technical replicates were performed by injecting 25 µg of digested product three times from the same sample vial into the LC–MS.