Vion ims qtof mass spectrometer

All measurements with the poly(dT) ladder were acquired on an ACQUITY UPLC I-Class system (Waters Corporation, Milford, MA, USA) coupled with a VION IMS qTOF mass spectrometer (Waters Corporation, Milford, MA, USA), measurements with the 24 nt thiolated oligonucleotide were acquired on a BioAccord LC-MS System with ACQUITY Premier (Waters Corporation, Milford, MA, USA). Source parameters were set as: 100 V source offset, 120 °C source temperature, 400 °C desolvation temperature, 800 L/h (L/h) desolvation gas flow and 50 L/h cone gas flow. The scan range of full MS was set to m/z 500.00–2000.00 at a scan rate of 1 Hz (1 s⁻¹). Data acquisition and processing were performed with UNIFI^TM (1.9.13.9 and 3.0.0.15). Calibration of MS data was achieved throughconstant infusion of leucine enkephalin calibration solution by Waters (SKU: 186006013) at a flow rate of 10 µL/min.

UPLC conditions are as follows: column, ACQUITY BEHC18 column (100 mm × 2.1 mm i.d., 1.7 µm; Waters, Milford, USA); solvent, the column was maintained at 45°C and separation was achieved using the following gradient: one to 30% B over 0–1 min, 30–60% B over 1–2.5 min, 60–90% B over 2.5–6.5 min, and 90–100% B over 6.5–8.5 min; the composition was held at 100 % B for 2.2 min, followed by 10.7–10.8 min, 100% to 1% B, and 10.8–13 min holding at 1% B at flow rate of 0.40 ml/min, where B is acetonitrile/methanol 2/3 (v/v)(0.1% (v/v) formic acid) and A is aqueous formic acid (0.1% (v/v) formic acid). Injection volume was 1 μl and the column temperature was set at 45°C.
The mass spectrometric data was collected using Waters VION IMS Q-TOF mass spectrometer equipped with an electrospray ionization (ESI) source operating in either positive or negative ion mode. The capillary voltages, DP, and CE were 2.5 kV, 40 V, and 6 eV, respectively. Source temperature and desolvation temperature were set at 115°C and 450°C, respectively, with desolvation gas flow at 900 l/h. Centroid data was collected from 50 to 1,000 m/z with scan time of 0.2 s and interscan delay of 0.02 s over a 13-min analysis time-period. The QCs were injected at regular intervals (every 10 samples) throughout the analytical run to provide the data set used for repeatability assessment.

LC–MS preparation was performed as previously described^{9 (link)}. Briefly, 100 mg of liver sample was homogenized with 20 µL internal standard (l-2-chloro-l-phenylalanine, 0.03 mg/mL; methanol configuration) and 800 µL methanol–water solution (4/1, v/v) before ultrasonic extraction for 5 min, incubation for 20 min at 4 °C, and centrifugation for 10 min at 14,000 g at 4 °C. Supernatant (200 µL) was transferred into a glass bottle for LC–MS metabolomics analysis. Supernatant underwent ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-Q-TOF/MS). Mass spectrometric data were collected using a Waters VION IMS Q-TOF mass spectrometer equipped with an electrospray ionization source operating in either positive or negative ion mode. Full details are provided in a previous study^{9 (link)}. Orthogonal partial least-squares discriminant analysis (OPLS-DA) was used to identify differential metabolites in MDD mice compared with CON mice.

Commercial reagents were purchased from Sigma-Aldrich, Aladdin Reagent Database Inc. and Bide pharmatech Ltd. (without further purification). ¹H, ¹³C NMR, and NOESY spectra were recorded on a Bruker AVANCE 600 instrument using DMSO-d₆ or CDCl₃ as a solvent. Chemical shifts δ are expressed in parts per million (ppm) and internally referenced to tetramethylsilane (TMS). The coupling constant J is reported in Hz. The following abbreviations are used: s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet. High-resolution mass spectra (HRMS) were obtained on a micrOTOF-QII mass spectrometer (Bruker, Germany) or a Vion^® IMS QTof mass spectrometer (Waters, UK). The ionization method chosen was electrospray ionization (ESI) operated in positive ion mode. All the reaction procedures were monitored by TLC using 40 precoated sheets of silica gel G/UV-254 of 0.25 mm thickness, Merck 60 F254 (BioLong, China). TLC plates were visualized by exposure to ultraviolet light and/or by exposure to iodine vapors, and the product was obtained by chromatography performed on silica gel (200–300 mesh).

UPLC–MS analysis was performed using an UPLC I-Class system with a BHC C18 column (100 mm  ×  2.1 mm, 1.7 μm) coupled to VION IMS QTOF mass spectrometer (Waters Corporation, Milford, MA, USA). The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B) was carried with elution gradient as follows: 0 min, 5% B; 3 min, 100% B; 4 min, 100% B; 4.1 min, 5% B; 6 min, 5% B, which was delivered at 0.4 mL/min. The column and autosampler were maintained at 35 and 10 °C, respectively. The injection volume was 2 μL. The ion source was operated in positive electrospray ionization (ESI) mode under the following specific conditions: capillary voltage, 1.0 kV; source temperature, 100 °C; desolvation gas temperature, 500 °C; desolvation gas flow, 800 L/h, and cone gas flow, 50 L/h. Nitrogen (> 99.5%) was employed as desolvation and cone gas. The scan range was from 50 to 1000 m/z. The scan time for each function was set to 0.2 s. The low collision energy was set at 6 eV, and the high collision energy was ramped from 20 to 45 eV. The data were acquired and processed using the MassLynx 4.1 software (Waters Co., Milford, USA) that was incorporated with the instrument.

After acquiring serum from the mouse, 80 μL serum was added to 240 μL cold methanol/acetonitrile (2:1, v/v). 10 μL internal tagging standard (L-2-chlorine-phenylalanine, 0.3 mg/mL, dissolved in methanol) was added, the samples were vortexed for 2 min, and then ultrasonic extraction was performed for 5 min. The samples were allowed to stand at -20° C for 20 min, and then centrifuged for 10 min (14,000 RPM, 4° C). 200 μL of the supernatant was loaded into a sample bottle with a lining tube for LC/MS analysis. A Waters UPLC I-class system equipped with a binary solvent delivery manager and sample manager, coupled to a Waters VION IMS Q-TOF Mass Spectrometer equipped with an electrospray interface (Waters Corporation, Milford, MA, USA) was used for LC/MS analysis. An Acquity BEH C18 column (100 mm × 2.1 mm; i.d., 1.7 μm; Waters, Milford, USA) was used for LC separation. Information on the peak picking, alignment, deconvolution, and further processing of raw LC-MS data can be found in previously published protocols [35 (link), 40 (link)] (Sah et al. 2017; Sonnerburg et al., 2016).