Vanquish ultra high performance liquid chromatograph

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)).

Sivelestat (0.1 g, H20203093, valid until 05/2022, Shanghai Huilun Jiangsu Pharmaceutical Co., Ltd.), Vanquish Ultra High-Performance Liquid Chromatograph (Thermo Fisher Scientific), Q Executive HFX High-Resolution Mass Spectrometer (Thermo Fisher Scientific), ultrasonicator (PS-60AL, Shenzhen Redbone Electronics Co., Ltd.), methanol and acetonitrile (CNW Technologies), ammonium acetate (SIGMA-ALDRICH), ammonium hydroxide (Fisher Chemical), ddH₂O (Watsons).