6545 esi q tof mass spectrometer

To determine the changes of metabolites in mice serum, 50 μL serum was mixed with 200 μL methanol, and centrifuged at 13,000 g for 15 min. The supernatant was extracted and filtered with a nylon membrane (22 μm). Sample separation was accomplished using a UPLC system of Agilent 1290 Infinity II series. The UPLC system was equipped with an Agilent Eclipse Plus C18 Rapid Resolution HD column (2.1 mm × 100 mm, 1.8 m). The mobile phases and gradient were set as described [31 (link)]. For MS acquisition, an Agilent 6545 ESI-Q-TOF mass spectrometer was employed. The mass spectrum parameters were the same as our previous publication [31 (link)]. Reference ions 112.985587 and 1033.988109 were used for real-time calibration during acquisition in negative ionization mode. Metabolites were identified with the METLIN Database (DB). Metabolites with DB scores above 80 and mass error lower than 5 ppm (0.0005%) were screened as biomarkers for statistical analysis. Metabolic pathway analysis was conducted using MetaboAnalyst 4.0 and KEGG online platform.

Analysis was performed on an Agilent 1290 Infinity HPLC coupled to an Agilent 6545 ESI Q-TOF mass spectrometer. MS was run in positive ionization mode, extended dynamic range (2 GHz), and standard mass range (m/z in the range of 300–3000 a.m.u.). The solvent mixtures used for LC-MS chromatography were: A = water + 0.1% formic acid (LC-MS grade), B = acetonitrile + 0.1% formic acid (LC-MS grade). The following conditions were used for peptide analysis. Column: Zorbax 300-SB C3 (5 µm, 150 × 2.1 mm, 300 Å silica); Flow Rate: 0.8 mL/min; Gradient: 1% B 0–2 min, linearly ramp from 1% B to 61% B 2 to 11 min, 61% B to 95% B 11 to 12 min. Post time is 1% B for 3 min. MS data were acquired from 4 to 11 min.