2.18. Untargeted metabolomic analysis

Plasma samples from 40 NSCLC patients were obtained before anti-PD-1 treatment. Untargeted metabolomic analysis were performed by Shanghai Applied Protein Technology Co., Ltd. (Shanghai, China). Briefly, to detect metabolites from plasma samples, 100 μL of each sample was added to pre-cooled methanol/acetonitrile/water solution (2:2:1, v/v/v), mixed by vortex, with 5 min incubation at 4 °C after each vortex step. After the final vortex step, the mixture was incubated on ice for 10 min. Then, 100 μL chilled HPLC-certified water was added to each sample and centrifuged at 13,000 × g for 10 min at 4 °C. Finally, the liquid phase (supernatant) of each sample was transferred into a new tube for UHPLC–QTOF-MS analysis. The data acquisition, principal component analysis and pathway impact analysis were performed by Shanghai Applied Protein Technology Co., Ltd.