Qe orbitrap ms

The total lipids of the different crabs were identified using a UHPLC system (Dionex, UltiMate, 3000 RSLC) coupled with QE Orbitrap/MS (ThermoFisher Scientific, Bremen, Germany). A BEH-HILIC column (100 × 1.0 mm, 1.7 μm, Sigma–Aldrich/Supelco, Bellefonte, PA, USA) was used for chromatographic separation. The flow rate was 0.1 mL /min, and the column temperature was 40 °C. Eluent A and eluent B were 5 mM ammonium formate in water and in acetonitrile, respectively. The chromatographic gradient elution mode was as follows: 0 min, 5% B; 4 min, 5% B; 10 min, 40% B; 15 min, 40% B; 16 min, 5% B; and 20 min, 5% B. The injection volume was 2 μL.

The metabolites from T. mesophilum SP-7-OMVs were extracted, and six parallel samples (labeled EV-1-1, EV-1-2, EV-1-3, EV-2-1, EV-2-2, and EV-2-3) were analyzed by ultrahigh-performance LC (UHPLC) coupled with Q Exactive (QE) Orbitrap/MS utilizing a nontargeting approach (Guangzhou Genedenovo Biotechnology Co., Ltd.). LC-tandem MS (MS/MS) analyses were performed by using a UHPLC system (1290, Agilent Technologies) with an Acquity HSS T3 column (2.1 × 100 mm; 1.7 μm) coupled with QE (Orbitrap MS, Thermo Fisher Scientific). The acquisition software (Xcalibur 4.0.27, Thermo Fisher Scientific) continuously evaluated the full-scan survey MS data as it collected and triggered the acquisition of MS/MS spectra depending on the preselected criteria. MS raw data (.raw) files were converted into the mzML format using ProteoWizard and processed via R package XCMS (version 3.2). OSI-SMMS (version 1.0, Dalian Chem Data Solution Information Technology Co., Ltd) was used for peak annotation after data processing with the in-house MS/MS database. Metabolites were matched with MS2 (the second-order mass spectrum), in which these metabolites could be defined as a “superclass”. Metabolites were mapped to the KEGG metabolic pathways for further shared pathway analysis (Supplementary material 1.1).

The analysis of extra- and intracellular metabolites was performed using an UPLC (Ultimate^TM 3000 RSLC system, Dionex Inc., Amsterdam, The Netherlands) with a QE Orbitrap MS (Thermo Fisher Scientific, Waltham, MA, USA) system equipped with an Xbridge amide column (4.6 × 100 mm, 3.5 µm, Waters, Milford, MA, USA). The following gradient was applied using 5 mM ammonium acetate (mobile phase A) and 100% ACN (mobile phase B) at a column flow of 0.30 mL/min and temperature of 30 °C: 0–3 min, 90 to 30% B; 3–12 min, 30 to 2%; 12.0–17.0 min, 2% B; 17.0–17.1 min, 2 to 90% B; and 17.1–23 min, 90% B, for re-equilibration. A 5 µL aliquot of the sample extract was injected into an injector port of an autosampler maintained at 5 °C during analysis. All analyses were performed in both ionization modes (negative and positive) based on the data-dependent MS/MS mode (m/z range: 60–900). The quality control (QC) samples obtained by mixing all intracellular extracts were injected every 10 runs [61 (link)]. The coefficient of variation (CV) and dilution factor of the QC sample were utilized to ensure analyte reproducibility and stability.