Untargeted Lipidomics Analysis With UHPLC-Q-Exactive Orbitrap MS

Lipid Extraction for Detection. A total of 20 mg lung tissue was used to extract lipids by the MTBE/MeOH/H2O system. First, tissue homogenate was extracted by 225 μl ice methanol solution containing internal standards (LPE (17:1), SM (17:0), TG (17:0/17:1/17:0) and PE (17:0/17:0), 5 μg/ml). After vortexing for 10 s, 750 μl MTBE was added, and the mixture was vortex for 10 min. Subsequently, 188 μl of deionized water was added and the mixture was vortexed for 20 s. After centrifuging 2 min by 14,000 rpm at 4 C, the upper phase was dried in a vacuum concentrator. Finally, the upper phase lipid was reconstituted in the mixed solvent of methanol: toluene (9:1, v/v) for lipidomic analysis.

Quality Control Samples. The preparation was carried out by mixing equal aliquots of 5 μl from each sample, and their pretreatment was carried out in the same manner as the samples. Five QCs were injected before samples, while one QC injection was inserted regularly after every 10 samples.

Chromatographic separation conditions. Untargeted lipidomics analysis of lung tissue was performed on an Ultra-high-performance liquid chromatography (Thermo Fisher Scientific, United States) coupled to hybrid Quadrupole-Exactive Orbitrap mass spectrometry (Thermo Fisher Scientific, United States). A reversed phase Waters Acquity UPLC CSH C18 (100 mm × 2.1 mm, 1.7 μm) maintained at 60°C was used for the chromatographic separation of lipids. The flow rate was set at 0.3 ml/min. Mobile phase A were ACN/H2O (6:4, v/v), and B IPA/ACN (9:1, v/v), both containing 10 mM ammonium formate and 0.1% formic acid for positive ionization mode. For the negative ionization mode, 10 mM ammonium acetate was used as buffer system. The elution gradient started with 15% B for 4 min, and then increased from 15 to 48% B in 1 min, from 48 to 82% B in 17 min, and from 82 to 99% B in 1 min, and maintained for 1 min, back to 15% B at 24.2 min and maintained for 5.8 min to equilibrate column.

Mass Spectrometer Conditions. The mass spectrometer was operated with the following parameters: spray voltage 3.5 kV (positive) and 3.0 kV (negative). For both ionization modes, the sheath gas and aux gas were separately maintained at 35 and 15 arbitrary units, while the capillary temperature and the heater temperature were 325 and 300°C, respectively. The MS/MS data was acquired by data dependent method and top 10 abundant ions were used for fragmentation. The normalized collision energy (NCE) was set 25, 35, and 45 eV, respectively.

Data processing of lipidomics. Data deconvolution, lipid identification and alignment were analyzed by MS-DIAL 3.3 (MS-DIAL software, Japan). The output data matrix was exported for further statistical analysis. MetaboAnalyst 4.0 (metaboanalyst.ca/faces/ModuleView.xhtml) was used for multivariate analysis. Principal component analysis and heatmap were performed for cluster and visulation. Furthermore, lipid metabolites enrichment analysis was based on ChemRICH (http://chemrich.fiehnlab.ucdavis.edu/) (Barupal and Fiehn, 2017). The differentially expressed lipids were screened using the Kruskal-Wallis test and Mann-Whitney test by R 3.6.3. Correlation analysis was conducted using Spearman correlation and visualized with Cytoscape 3.8.2 (Cytoscape software, United States).