Surveyor plus hplc

The concentration of amines in caecal digesta was determined using the HPLC method described previously [17 (link)]. The digesta samples were homogenised in ultra-pure water by intensive vortexing and supernatants were obtained by centrifugation at 10,000 rpm for 15 min. The supernatant was subsequently diluted five-fold with an acetone and water mixture (2:1) and alkalised with borax buffer (0.1 M, pH 10.5). Heptylamine was then added as an internal standard to a final concentration of 5 μg/mL. Subsequently, amines were derivatised by incubation with 1% dansyl chloride in acetone for 25 min at 65 °C in a water bath in the dark and then extracted using Waters SEP-PAK serif™ C18 cartridges for solid phase extraction (6 mL, 500 mg; Waters, Watford, Hertfordshire, UK). Separation was carried out using a Finnigan Surveyor Plus HPLC (Thermo Scientific, San Jose, CA, USA) with a photodiode array detector and a Waters Symmetry Shield RP18 column (150 × 3.9 mm i.d., 5 μm) preceded by a guard column (Waters Symmetry Shield RP18, 20 × 3.9 mm, 5 μm). The mobile phase was composed of 5% acetonitrile in water and 100% acetonitrile, flowing under a gradient elution. Dansyl derivatives of amines were detected by measuring absorbance at 254 nm, and their concentrations were determined from standard curves.

High-resolution tandem mass spectrometry (HR-MS/MS) was carried out using a Finnigan LTQ Orbitrap coupled to a Surveyor Plus HPLC pump and autosampler (Thermo Fisher, Bremen, Germany) in positive ionization mode using an MS range of m/z 100–2000, an MS2 range of m/z 200–1500, and an MSn range of m/z 0–1000, with 30,000 resolution. LC-MS data were acquired using Xcalibur version 2.2. HR-MS/MS raw data files were converted from .RAW to .mzXML format using the Trans-Proteomic pipeline (Institute for Systems biology, Seattle) [29 (link)], and clustered with MS-Cluster using Global Natural Products Social Molecular Networking (GNPS-https://gnps.ucsd.edu) [30 (link)]. A molecular network was created using the online workflow at GNPS. The following settings were used for generation of the network: minimum pairs cos 0.6; parent mass tolerance, 1.0 Da; ion tolerance, 0.2; network topK, 10; minimum matched peaks, 6; minimum cluster size, 2. Data were visualized and analyzed using Cytoscape 3.6.0.
The compounds common to both liquid and solid growth conditions of E. chevalieri MUT 2316 were highlighted, as well as the compounds unique to each condition.