Uplc hss t3 column

1 μg of genomic DNA was incubated with 5 U of DNA Degradase Plus (Zymo Research) at 37°C for 3 h and filtered through Amicon 10 kDa centrifugal filter units (Millipore). The concentrations of 2′-deoxycytidine, 5-methyl-2′-deoxycytidine and 5-hydroxymethyl-2′-deoxycytidine in the filtrate were determined using an AB Sciex Triple Quad 6500 mass spectrometer fitted with an Agilent Infinity 1290 LC system and an Acquity UPLC HSS T3 column. The global levels of mC and hmC were expressed as percentages over total 2′-deoxycytidines.

The DAN was characterized by the following methods chemically: 20 mg sample was weighted into an EP tube accurately, and 1 mL extract solution (water:methanol:acetonitrile = 1:2:2) containing 1 μg/mL internal standard was added. The samples were homogenized at 35 Hz for 4 min and sonicated for 5 min with a 3 times repetition in an ice-water bath. Then all samples were incubated for 1 h and centrifuged at 12,000 rpm for 15 min. The last supernatant fluid was transferred into a fresh glass vial for LC/MS analysis. The analysis was performed by a UHPLC system (Agilent Technologies, Santa Clara, CA, USA) with a UPLC HSS T3 column connected to a QE mass spectrometer (Santa Clara, CA, USA). The injection volume was 2 μL. The QE mass spectrometer was used because it can acquire MS/MS spectra in information-dependent acquisition (IDA) mode under the control of the acquisition software (Xcalibur 4.0.27, Thermo, Waltham, MA, USA). In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The ESI source conditions were set as follows: Aux gas flow rate was 15Arb, sheath gas flow rate was 45 Arb, capillary temperature was 400 °C, MS/MS resolution was 17,500, full MS resolution was 70,000, spray voltage was 4.0 kV (positive) or −3.6 kV (negative), and collision energy was 20/40/60 in NCE mode, respectively.

A total of 50 μL of serum and approximately 10mg of hippocampal tissues (wet weight) were used for sample preparation following previously reported methods (51 (link), 52 (link)). Samples were analyzed using an Agilent 6550 iFunnel Q-TOF LC/MS system (Agilent) with an Acquity UPLC HSS T3 column (53 (link)). Raw data were converted to mzXML format by ProteoWizard 3.0 package and uploaded to XCMS online (54 (link)). Metabolites were identified against METLIN (55 (link)) and Human Metabolome Database (56 (link)). Multivariate analysis was performed with SIMCA 13.0 (Umetrics). VIP scores were assessed. Discriminated metabolites were defined with a VIP > 1.0 by partial least squares-discriminant analysis (PLS-DA), P < 0.01, FDR-adjusted P < 0.05. Pathway enrichment analysis were analyzed using Metaboanalyst 4.0 (57 (link)).