Hss t3 c18 column

Metabolomics data were acquired via previously published UHPLC-HRMS methods [26 (link),32 (link),33 (link),34 (link),35 (link),36 (link),37 (link)]. The analysis utilized a Vanquish UHPLC system coupled to a Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an HSS T3 C18 column (2.1 mm × 100 mm, 1.7 µm, Waters Corporation) held at 50 °C. A binary pump was used with water + 0.1% formic acid (A) and methanol + 0.1% formic acid (B) as mobile phases. The mobile phase gradient started from 2% B, increased to 100% B in 16 min, and was then held for 4 min with a flow rate of 400 µL/min. Mass spectral data were collected using a data-dependent acquisition mode in positive polarity at 70–1050 m/z. QCSP and blank injections were placed at a rate of 10% throughout the study samples. An injection volume of 5 µL was used for analysis of each sample. Raw UHPLC-HRMS data were imported into Progenesis QI (version 2.1, Waters Corporation, MA, USA) for alignment, peak picking, and deconvolution. Background signals were removed by filtering out peaks with a higher average abundance in the blank injections as compared to the QCSP injections. Data were normalized using a QCSP reference sample using the “normalize to all” function in progenesis [38 (link)].

LC-MS setup: The injection volume was 10 μL. The separation was performed on an HSS T3 C18 column (100 mm × 2.1 mm × 1.8 μm, Waters) with the column temperature maintained at 50 °C. The gradient elution program was set as follows: 0-2 min, 100% A; 2-11 min, 0%-100% B; 11-13 min, 100% B; 13-15 min, 0%-100% A. The Q-TOF mass spectrometer was operated in positive and negative ion modes. ESI source temperature: 120°C; desorption temperature: 450°C; desorption gas: 800 L/h; cone gas: 50 L/h; TOF mass range: 50-1200 Da; scan time: 0.2s. The LC-MS metabolic profiles were acquired using an ACQUITY UHPLC system (Waters Corporation Milford, USA) with an AB SCIEX Triple TOF 5600 system (AB SCIEX, Framingham, MA, USA) in the ESI positive and negative ion modes. The QC samples were injected at regular intervals (every 6 samples) throughout the analysis to provide a data set to assess reproducibility. The raw LC-MS data were provided by Luminous (Shanghai, China), and metabolites were identified primarily based on RT m/z pairs and tandem mass spectrometry (MS/MS) spectra, HMDB (https://hmdb.ca/), LIPID MAPS (https://lipidmaps.org/), and a self-constructed database. The data matrix, including 3D datasets of m/z, RT peaks, and intensities, was exported as an Excel file for further analysis.

A Waters UHPLC coupled in tandem to a Waters SYNAPT G1 HDMS mass spectrometer was used to generate accurate mass data. Optimization of the chromatographic separation was done utilizing a Waters HSS T3 C18 column (150 mm × 2.1 mm, 1.8 μm) and the column temperature controlled at 60°C. A binary solvent mixture was used consisting of water (Eluent A) containing 10 mM formic acid (natural pH of 2.3) and acetonitrile (Eluent B) containing 10 mM formic acid. The initial conditions were 90% A at a flow rate of 0.4 mL min^–1 and were maintained for 1 min, followed by a linear gradient to 1% A at 35 min. The conditions were kept constant for 2 min and then changed to the initial conditions. The runtime was 40 min and the injection volume was 1 μL. Samples were kept at 6°C in the Sample Manager during the analysis.

Briefly, BAs in 100 mg of fecal samples were extracted using 1 mL of methanol. BAs in the optioned supernatant after 30 min of centrifugation at 12,000 rpm were analyzed. BAs were separated using a Dionex™ UltiMate™ 3000 Rapid Separation LC (RSLC) system (Thermo Scientific, Waltham, MA, USA) equipped with an HSS T3C18 column (2.1 mm × 100 mm, 1.8 μm, Waters, Milford, CT, USA). The injection volume, temperature, and flow rate were set to 50 °C, 10 μL, and 300 μL/min, respectively. Methanol (A) and 2 mmol/L of ammonium acetate (B) were used as the mobile phases. Furthermore, the mass spectrometry was performed using a Thermo Scientific TM Q Exactive TM hybrid quadrupole Orbitrap mass spectrometer equipped with a HESI-II probe. The negative HESI-II spray voltage was 3.5 kV. The heated capillary temperature, sheath gas pressure, auxiliary gas setting, and heated vaporizer temperature were 320 °C, 30 psi, 10 psi, and 300 °C, respectively. The auxiliary gas, sheath gas, and collision gas were all nitrogen at 1.5 m Torr. The full mass scan parameters were set as follows: an auto gain control target under 1 × 10⁶, a resolution of 70,000, an m/z range of 150–1500, and a maximum isolation time of 50 ms [19 (link)].

The concentration of 147 in the ER was determined by treating HEK293T cells for 15 min with 10 µM 147. The cells were scraped off the plate with 1 mM EDTA in TBS and the cellular pellet was lysed with 0.1% digitonin in HEPES (pH 7.5), 100 mM NaCl. The supernatant (cytosol) was analyzed for enrichment of cytosolic proteins (HSC70) and the pellet (ER) was analyzed for ER proteins (ERdj3) by immunoblot. To determine the concentrations of 147 in the ER and cytosol, each enriched fraction was subject to a chloroform: methanol extraction by adding 4× volume of both chloroform and methanol to precipitate the proteins. The organic phase was collected, concentrated, and resuspended in methanol. 147 concentrations were determined using a Waters TQ-XS triple quad mass spectrometer by injecting 5 µL of sample onto a Waters HSS T3 C18 column (2.1 × 10 mm, 1.8 µM) with a flow rate of 0.3 mL per minute and comparing the observed peak areas to a calibration curve.

Metabolomics data were acquired on a Vanquish UHPLC system coupled to a QExactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (ThermoFisher Scientific, San Jose, CA, USA), as described previously [69 (link)]. Our UPLC–MS reversed phase platform was established based on published methods [70 (link),71 (link)]. Metabolites were separated using an HSS T3 C18 column (2.1 mm × 100 mm, 1.7 μm, Waters Corporation, Milford, MA, USA) at 50 °C with binary mobile phase of water (A) and methanol (B), each containing 0.1% formic acid (v/v). The UHPLC linear gradient started from 2% B, and increased to 100% B in 16 min, then held for 4 min, with the flow rate at 400 μL/min. The untargeted data were acquired in positive mode from 70 to 1050 m/z using the data-dependent acquisition mode.

A Waters Classic UPLC, coupled in series to a Waters SYNAPT G1 HDMS mass spectrometer was used to generate full scan accurate mass data. Optimization of the chromatographic separation was done utilizing a Waters HSS T3 C18 column (150 mm × 2.1 mm, 1.8 µm) and the column temperature controlled at 60°C. A binary solvent mixture was used consisting of water (Eluent A) containing 10 mM formic acid (natural pH of 2.4) and acetonitrile (Eluent B) containing 10 mM formic acid. The initial conditions were 100% A at a flow rate of 0.4 ml/min and were maintained for 1 min, followed by a linear gradient to 1% A at 15 min. These conditions were kept constant for 2 min and then changed to the initial conditions. The runtime was 20 min, and the injection volume was 1 µl. Samples were kept cool at 6°C in the Waters Sample Manager during the analysis.