Orbitrap exactive

All of the commercially available reagents, catalysts, bases, and solvents were used as purchased without further purification. Starting materials and reaction products were purified by flash chromatography using SiO₂ as a stationary phase, eluting with n-hexane/ethyl acetate mixtures. ¹H NMR (400.13 MHz), ¹³C NMR (100.6 MHz), and ¹⁹F spectra (376.5 MHz) were recorded with a Bruker Avance 400 spectrometer. Splitting patterns are designed as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or bs (broad singlet). HRMS of samples were recorded on an Orbitrap Exactive (Thermo Fisher, Waltham, MA, USA). Melting points were determined with a Büchi B-545 apparatus and are uncorrected.

HRMS analysis was performed by ionization (ESI) mass spectrometry (MS). The extracts were dissolved to a final concentration of 1-2 pmol/μL in methanol. Compounds were measured in negative and positive modes by full mass scanning (m/z 50 to 1000) using a Thermo Scientific Orbitrap Exactive mass spectrometer equipped with a heated electrospray ionization source (HESI-II). The instrument parameter settings are as follows: sheath gas 10 in positive mode and 20 in negative mode (arbitrary units), spray voltage 3.5 kV and 3 kV in positive and negative mode, respectively, with a 275°C capillary temperature. Mass spectra were collected at a resolution of 100,000. Data processing was performed using the associated software, Xcalibur 2.2 and Exactive 1.1.

For identification of R. jostii RHA1 γ-butyrolactone-like molecules, HPLC-MS analysis was performed using an Accella1250™ HPLC system coupled with the benchtop ESI-MS Orbitrap Exactive™ (Thermo Fisher Scientific, San Jose, CA). A Reversed Phase C18 (Shim Pack Shimadzu XR-ODS 3 × 75 mm) column was used and a gradient from 2% to 95% of acetonitrile:water (0.1% Formic Acid) as follows: 2 min 2% acetonitrile, 2–10 min gradient to 95% acetonitrile, 1 min 95% acetonitrile. To separate further the peaks from A-factor and 6-dehydro SCB2, a gradient from 2% to 80% acetonitrile was applied to the separation: 2 min 2% acetonitrile, 2–25 min in 2–80% acetonitrile, 1 min 80% acetonitrile. Data was analysed using Xcalibur software from Thermo Scientific. LC-MS analysis was performed with 2–4 biological replicates per strain.

The prepared samples were applied on an analytical system consisting of ultra-high performance liquid chromatography (UHPLC) Ultimate 3000 (Thermo-Fisher Scientific, Waltham, MA, USA) connected with a high-resolution accurate mass spectrometer (UHPLC/HRAM-MS, Orbitrap Exactive, Thermo-Fisher Scientific, Waltham, MA, USA). Methanol (100%) and water with 5 mM ammonium formate were used as mobile phases for gradient elution. The gradient began at 10% (0 min to 2 min) and then grew to 100% of A in 10 min, which was maintained until 15 min for washing of column, and finally was returned to initial conditions (10% A at 16 min) and kept for 4 min for system conditioning. The separation was performed using Kinetex Synergi Hydro-RP column (2.5 µm, 100 × 2.1 mm, Phenomenex, Torrance, CA, USA) tempered to 35°C and the flow rate set to 250 µL/min. The injection volume was 5 µL. The MS analysis was performed using positive ESI ionization and data was collected in FullMS/AIF scanning mode (resolution of 70,000 FWHM, m/z range from 170 to 900).

LC–MS analysis was performed as described in [30 (link), 49 (link)]. Metabolites were extracted from supernatant media before and after treatment and by lysing cells in ice-cold methanol/acetonitrile/H₂O (50:30:20). Samples were shaken at 4 °C for 10 min and then centrifuged for 15 min at 16,000 g, and the supernatant was collected and analyzed by LC–MS. Analytes were separated using hydrophilic interaction liquid chromatography with a SeQuant ZIC-pHILIC column (2.1 3 150 mm, 5 mm) (Merck) and detected with high-resolution, accurate-mass mass spectrometry using an Orbitrap Exactive in line with an Accela autosampler and an Accela 600 pump (Thermo Scientific). The elution buffers were acetonitrile for buffer A and 20 mM (NH₄)₂CO₃ and 0.1% NH₄OH in H₂O for buffer B. A linear gradient was programmed starting from 80% buffer A and ending at 20% buffer A after 20 min, followed by wash (20% buffer A) and re-equilibration (80% buffer A) steps with a flow rate of 100 ml/min. The mass spectrometer was fitted with an electrospray-ionization probe and operated in full-scan and polar-switching mode with the positive voltage at 4.5 kV and negative voltage at 3.5 kV. Metabolite identification and data analysis were carried out using LCQUAN software (Thermo Scientific).

MSCs were stimulated with nanovibration for 7 and 14 days in 2D and 3D (collagen gels; 2 mg ml⁻¹) culture. Nonstimulated samples cultured in expansion media and OGM were used as controls. Metabolites were extracted using a 1:3:1 chloroform/methanol/water extraction buffer and vigorously shaken at 4°C for 1 hour. Following this, metabolite extraction solution was collected, transferred to 1.5-ml tubes, and centrifuged for 3 min at 13,000g at 4°C. Metabolomics was performed through hydrophilic interaction LC-MS analysis (UltiMate 3000 RSLC, Thermo Fisher Scientific) with a 150 mm by 4.6 mm ZIC-pHILIC column running at 300 μl min⁻¹and Orbitrap Exactive (Thermo Fisher Scientific). A standard pipeline, consisting of XCMS (58 (link)) (peak picking), MzMatch (59 (link)) (filtering and grouping), and IDEOM (60 (link)) (further filtering, postprocessing, and identification), was used to process the raw mass spectrometry data. Identified core metabolites were validated against a panel of unambiguous standards by mass and retention time. Further putative identifications were allotted mass and predicted retention time (22 (link)). Means and SEs of the mean were generated for every group of picked peaks, and the resulting metabolomics data were uploaded to IPA software for pathway analysis.