Acquity uplc beh amide

Bacterial supernatant samples were processed and untargeted metabolic profiling of bacterial supernatant was performed by liquid chromatography-mass spectrometry (LC-MS) as described previously (Zou et al., 2013 (link)). Briefly, metabolite samples were extracted from the supernatant with methanol and acetonitrile combined with isotope-labeled compounds. Samples were detected by Vanquish Ultra-high performance liquid chromatograph (Thermo Fisher Scientific) and separated by Waters ACQUITY UPLC BEH Amide liquid chromatography. Data were collected with a Thermo Q Exactive HFX mass spectrometer (Thermo Fisher Scientific). Original data was managed as follows: removing deviation value and missing value, filling missing value, and normalization (Dunn et al., 2011 (link)). Then performing hierarchical clustering analysis and principal component analysis (Ringnér, 2008 (link)). Differential metabolites were screened through variable importance in projection (VIP) value combined with P value and fold change. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was used for annotation of the different metabolites as described previously (Ogata et al., 1999 (link)).

The metabolites in serum and liver were analyzed by extraction, computer detection and statistical analysis. First, 25 mg liver was added to 500 μL of extract solution (methanol:acetonitrile:water = 2:2:1) and 100 μL of serum sample was added to 400 μL of extract solution (methanol:acetonitrile = 1:1), both of which contained the isotopically labeled internal standard mixture. Mix well and sonicate for 10 min under ice-water bath conditions, noting that the liver needs to be processed by grinding. The supernatant solution was obtained by centrifugation at 12,000 rpm for 15 min at 4°C after 1 hour at −40°C. The target compounds were chromatographically separated on a Waters ACQUITY UPLC BEH Amide (2.1 mm × 100 mm, 1.7 μm) column using a Vanquish (Thermo Fisher Scientific) ultra-performance liquid chromatograph. The A-phase of liquid chromatography was aqueous containing 25 mmol/L ammonium acetate and 25 mmol/L ammonia, and the B-phase was acetonitrile. The data collection was performed on a QE HFX mass spectrometer (Orbitrap MS, Thermo) under the control of acquisition software (Xcalibur, Thermo). The raw data were also processed using ProteoWizard software.

Metabolite extraction was performed in strict accordance with the operating instructions, followed by onboard detection.
On-Board Detection. The target compounds were chromatographed on a Waters ACQUITY UPLCBEH Amide (2.1 mm × 100 mm, 1.7 μm) column using a Vanquish (Thermo Fisher Scientific) ultra-performance liquid chromatography. The A phase of the liquid chromatography was aqueous containing 25 mmol/L ammonium acetate and 25 mmol/L ammonia, and the B phase was acetonitrile. The sample tray temperature is 4°C, and injection volume is 2 μL.
The Thermo Q Exactive HFX mass spectrometer was capable of primary and secondary mass spectrometry data acquisition under the control of the control software Xcalibur (Thermo). Detailed parameters were as follows: sheath gas flow rate, 30 Arb; Aux gas flow rate, 25 Arb; capillary temperature: 350°C; full ms resolution, 60000; MS/MS resolution, 7500; collision energy, 10/30/60 in NCE mode; and spray voltage: 3.6 kV (positive) or −3.2 kV (negative).
Data Processing. The raw data were converted to mzXML format by ProteoWizard software, and then the peak identification, peak extraction, peak alignment, and integration were performed using the R package (kernel XCMS) written by ourselves and then matched with the BiotreeDB (V2.1) self-built secondary mass spectrometry database for substance annotation.