Xevo qtof

Peptide identification was done by running tandem MS/MS experiments using a 3%–35% B gradient over 120 min with a Xevo QTOF (Waters). This was supplemented with a 20 min gradient separation to identify and correct the retention time for all samples. The MS tolerance was set to 3 ppm with a MS/MS tolerance at 0.1 Da. The resulting MS/MS datasets were analysed with the Mascot search within Mascot distiller (Matrix Science). All peptides with a Mascot score >15 were analysed using HD-Examiner Software (Sierra Analytics). The full list of peptides was then manually validated by searching a non-deuterated protein sample's MS scan to test for the correct m/z state and check for the presence of overlapping peptides. Ambiguously identified peptides were excluded from all subsequent analysis. The first round of analysis and identification were performed automatically by the HD-Examiner software, but all peptides (deuterated and non-deuterated) were manually verified at every state and time point for the correct charge state, m/z range, presence of overlapping peptides and the expected retention time. All HDX–MS results are presented as relative levels of deuterium incorporation and no correction for back exchange is applied, because no fully deuterated protein sample could be obtained.

The procedure of phenolic compounds' extraction was acquired from the protocol previously published [25 (link)] using solid-phase C8 cartridges. Then, the hydrophilic compounds were separated on a C18 UPLC column (BEH Acquity, 100 mm × 2.1 mm × 1.7 μm, Waters, USA) connected to an Acquity UPLC system equipped with PDA 200–500 nm and mass spectrometer Xevo-Q-TOF (Waters, Milford, CT, USA). The mobile phase was a mixture of A—deionized waterformic acid (99.5 : 0.5), and B—acetonitrileformic acid (99.5 : 0.5), at a flow rate of 0.5 ml/min. The electrospray ionization source of the mass spectrometer operated at a capillary voltage of ±2.80 kV in the positive and negative ion mode, respectively, a sampling cone of 66 kV, and an extraction cone of 4.0 kV. Collision energy was set at 0, 20, 20–30, 30, and 30–50 kV. Data were processed using MassLynx 2.0 (Waters, Milford, CT, USA). Single components were identified by comparison of experimental mass, UV absorption spectra, and retention time with standards and literature data.

The molecular masses of biosurfactants were determined by electrospray ionization mass spectrometry (ESI-MS) with a Xevo Q-TOF (Waters, Milford, MA, USA) mass spectrometer. Samples were suspended in 50% ACN/0.2% formic acid (v/v). LC-MS mass spectra were performed, in positive mode for lipopeptides and negative mode for rhamnolipids, with a cone voltage ramping from 20 to 40 V. The spray voltage was set to 3.0 kV, the source temperature to 120°C and the desolvation temperature to 450°C. The LC separation was conducted using the same column and gradient as for HPLC analyses indicated previously except for the flow rate which was reduced to 0.5 ml min⁻¹. LC-MS/MS mass spectra were performed in the MS^E mode (Waters, Milford, MA, USA). Briefly, in the MS^E mode, mass spectrometric scans alternate all along the experiment between low (10 V) and high (ramping from 30 to 60 V) fragmentation energy delivering for the same LC separation two chromatograms corresponding to MS and MS/MS analyses, respectively.

Optical rotations were measured using an ATAGO AP-300 automatic polarimeter (Saitama, Japan), and the high-resolution mass spectra (HRESI-TOFMS) were obtained on a Waters Xevo Q-TOF direct probe/MS system using ESI⁺ mode and microchannel plates MCPs detector (Milford, MA, USA). In contrast, the IR spectra were measured on a One Perkin Elmer infrared-100 (Shelton, CT, USA). The NMR data were recorded on a JEOL ECZ-500 spectrometer (Tokyo, Japan) at 500 MHz for ¹H and 125 MHz for ¹³C using TMS as the internal standard. Chromatographic separations were conducted on a silica gel G60 (Merck, Darmstadt, Germany, 70–230 and 230–400 mesh), and the TLC plates were precoated with GF₂₅₄ (Merck, 0.25 mm), after which detection was performed by spraying with 10% H₂SO₄ in ethanol, before heating.

The composition of propolis extracts was analysed by the Waters Acquity UPLC system (Waters, Milford, CT, USA) equipped with PDA 200–500 nm, a mass spectrometer Xevo-Q-TOF (Waters, Milford, CT, USA), and a column BEH C18 130 Å (1.7 μm, 2.1 mm × 150 mm) (Waters, Milford, CT, USA). Analyses were performed according to previously described methods [15 (link),16 (link)].

The determination of phenolic compounds from the oils was preceded by the isolation of phenolic compounds from the oils by solid-phase extraction (SPE) based on the method described previously by Pirisi et al. [36 (link)], followed by identification and quantitative analysis of these compounds by ultra-pressure liquid chromatographic analysis. Analysis was performed using a Waters Acquity UPLC system (Waters Corporation, Milford, CT, USA) connected to a PDA detector 200–500 nm and mass spectrometer Xevo-Q-TOF (Waters, Milford, CT, USA). Separation was performed, as in the case of tocochromanol and carotenoid determinations, on a C18 column with the same parameters. The injection volume was 5 µL. Solvent mixtures A and B were used for separation. Mixture A contained 99.5% water and 0.5% formic acid (v/v). Mixture B was 99.5% acetonitrile and 0.5% formic acid (v/v). The flow rate of the mobile phase was 0.6 mL/min. Chromatograms were recorded at wavelengths of 280 and 320 nm [35 (link)].

Mass spectrometric analyses were performed with a Xevo Q-TOF instrument equipped with an electrospray ionisation source (ESI) (Waters Corporation). ESI conditions were as follows: capillary voltage 2.6 kV, sampling cone voltage 40 V, source temperature 120 °C, desolvation temperature 300 °C, desolvation N₂ gas flow 600 L/h, collision energy was changed between 10 eV and 45 eV, depending on the structure analyzed. High purity nitrogen was used as the collision gas. Full-scan mass spectra were acquired in the range of m/z 100–2000 in negative ionisation mode. Masslynx 4.1 software was used for data acquisition and qualitative analysis.