Triple tof 5600 mass spectrometer

Compared with the phenolics contents in the supernatant of initial cultures (0 h), the supernatants of cultures with significant change (p < 0.05) in phenolics contents after fermentation for 48 h, were subjected for qualitative analysis and follow-up analysis.
Compound identification was achieved by UHPLC-ESI-QTOF-MS/MS as reported previously by our team (18 (link)). The analysis was performed with an Agilent 1290 UHPLC system coupled to a Triple-TOF 5600+ mass spectrometer (Agilent Technologies, California, USA) and equipped with an Agilent EclipsePlus C18 column (2.1 × 100 mm, 1.8 μm, Palo Alto, California, USA) and ESI source operating in negative ionization mode. The parameters set were: capillary voltage, 4,500 V; flow rate, 0.4 ml/min; column temperature 35°C; ion source temperature 500°C; and injection volume, 4 μl. The mobile phase consisted of 0.4% formic acid in water (A) and acetonitrile (B). The analysis was carried out with a gradient elution as follows: 0–16 min, 5–25% B; 16–18 min, 25–35% B; 18–20 min, 35–50% B. The abundance of ions (100–1,000 m/z) was scanned. Confirmation was obtained by comparison with external standards whenever available and by consulting the phytochemical dictionary of natural products database (DNP).

All fractions were resuspended in 20 µl buffer A (2% acetonitrile, 0.1% formic acid) and peptide concentration was measured using the 280 nm NanoDrop assay. One microgram of total peptide was analysed in DDA mode on an Agilent 1260 HPLC coupled to a TripleTOF 5600+ mass spectrometer equipped with NanoSource III. Each fraction was spiked with 0.1 µl of iRT calibration mix (Biognosys, AG) and loaded onto a 0.3×5 mm ZORBAX C18 (Agilent Technologies) trap column. Peptides were separated on a 75 µm×15 cm analytical column packed with Reprosil Pur C18AQ beads, 3 µm, 120 Å (Dr. Maisch, GmbH) with a manually pulled integrated spraying tip. A linear gradient of 2-40% buffer B (98% acetonitrile, 0.1% formic acid) in 90 min and flow rate of 250 nl/min was used for peptide separation. All data were measured in positive mode. Full profile MS scans were acquired in the m/z mass range of 340-1500 with 250 ms filling time; MS/MS scans for the 20 most intense ions with charge state from 2+ to 5+ were acquired in m/z mass range of 280-1500 with 100 ms filling time. Dynamic exclusion of fragmented ions was set to 12 s.

Metabolic analysis was performed using APTbiotech (Shanghai, China), with 10 replicates for each group (HT29-shNFIB or control). Briefly, 5×10⁷ cells were processed using an ultrasonic homogenizer in a mixture of methanol, acetonitrile, and water (2:2:1, vol/vol). The dried supernatant was subjected to Liquid Chromatography-Mass Spectrometry (LC-MS) analysis. The samples were separated by Agilent 1290 Infinity LC system and detected using a Triple TOF 5600 mass spectrometer. The obtained raw data were transferred to mzXML format via ProteoWizard. The data were then imported into the software XCMS, which performs peak detection, peak identification, and peak alignment. Variable Importance of Projection (VIP) values obtained from the OPLS-DA model was used to rank each variable’s overall contribution to group discrimination. Differential metabolites were selected with VIP values greater than 1.0 and p-values less than 0.05.