Analysis of serum metabolome by liquid chromatography-mass spectrometry (LC-MS)
SETTLE
One hundred µl liquid samples were accurately weighed, and the metabolites were extracted using a 400-µL methanol:water (4:1, vol/vol) solution. The mixture was allowed to settle at −20°C and treated by high throughput tissue crusher Wonbio-96c (Shanghai Wanbo Biotechnology Co., Ltd.) at 50 Hz for 6 min, followed by vortex for 30 s and ultrasound at 40 kHz for 30 min at 5°C. The samples were placed at −20°C for 30 min to precipitate proteins. After centrifugation at 13000g at 4°C for 15 min, the supernatant was carefully transferred to sample vials for LC-MS/MS analysis.After LC-MS/MS analyses, the raw data were imported into the Progenesis QI 2.3 (Nonlinear Dynamics, Waters, USA) for peak detection and alignment. Metabolic features detecting at least 80% in any set of samples were retained. After filtering, each metabolic feature was normalized by sum. The internal standard was used for data quality control (QC) (reproducibility). Metabolic features with a relative standard deviation (RSD) of QC of > 30% were discarded. Mass spectra of these metabolic features were identified by using the accurate mass, MS/MS fragments spectra, and isotope ratio difference by searching in reliable biochemical databases, such as the Human Metabolome Database (HMDB) (The preprocessed data were analyzed on the Meggie Biocloud platform (