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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14030/m_MTBLS14030_LC-MS_alternating_hilic_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14030/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14030/a_MTBLS14030_LC-MS_alternating_hilic_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14030/s_MTBLS14030.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14030TraceFinder 5.1 (Thermo Scientific) was used to quantify the targeted metabolites by area under the curve using accurate mass measurements (±5 ppm) and prior established retention time as determined with pure standards. Metabolite isotopologue distributions were corrected for naturally occurring 13C abundance and tracer impurity using the IsoCorrectoR package. MetaboLightsPublicLiquid Chromatography MS - alternating - hilicLC-MS analysis was performed with an Vanquish Horizon UHPLC system (ThermoFisher Scientific, San Jose, CA). Dried metabolite extracts were re-suspended in 100 μL LC-MS grade water (W6212, Fisher Scientific, USA). A 5 μL aliquot was separated on a Luna 3μm NH2 100A (150 x 2.0mm, 00F-4377-B0, Phenomenex) with a flow rate of 0.3 mL/min for a total duration of 40 min. The gradient was composed of 85% solvent B (7 min), 85-5% B (18 min), 5% B (7.9 min), 5-85% B (0.1 min) and followed by a 7 min equilibration of 85% B; solvent A: 5 mM ammonium acetate (NH4AcO,pH 9.9; 14267-25G, Fluka, USA); solvent B: 100% acetonitrile (ACN; A9554, Fisher Scientific, USA).13C-glucose tracing analysis of IFN-treated A549 cells expressing wild-type SARS-CoV-2 Nsp3-4 or a deISGylation-defective Nsp3-4 mutant protein.CaltechTing-Yu WangA-549 cellmass spectrometry assayMetabolites were extracted by adding 1 mL ice-cold 80% methanol and by incubating the cells at −80 °C for 20 min. Cells were scraped, transferred to microcentrifuge tubes, and centrifuged at maximum speed for 5 min at 4 °C. The supernatant was collected, while the pellet was re-extracted with 200 µL 80% methanol and centrifuged again. Supernatants for each sample were combined and dried in a vacuum concentrator without heating. Dried metabolite extracts were stored at −80 °C before LC-MS analysis.Homo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS14030Michaela Gack. Florida Research and Innovation Center, Cleveland Clinic, Port Saint Lucie, USA. GACKM@ccf.org.Junji Zhu. Florida Research and Innovation Center, Cleveland Clinic, Port Saint Lucie, USA. ZHUJ6@ccf.org.Ting-Yu Wang. Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA, USA. tywang@caltech.edu.The RAW data was directly imported to TraceFinder 5.1 (Thermo Scientific) to quantify the targeted metabolites by area under the curve using accurate mass measurements (±5 ppm) and prior established retention time as determined with pure standards. TreatmentTransfectiontywang@caltech.eduA549 cells (~1 × 106 cells per sample) cultured in DMEM containing 10% FBS were transfected for 24 h with either empty vector or HA-tagged Nsp3-4 WT or PLpro deISGylation-deficient mutant (Nsp3-4-Mut), followed by mock treatment or stimulation with IFNα (500 U/mL) for an additional 24 h. For isotope tracing, cells were incubated in 13C-glucose-containing DMEM medium for 15 min. MetabolomicsThermo Scientific Vanquish UHPLC SystemOrbitrap EclipseA-549 cellliquid chromatography-mass spectrometryuntargeted analysisHomo sapienstargeted metabolite profilingU-13C glucose tracingexperimental sampleThermo Scientific Vanquish UHPLC SystemOrbitrap EclipseA-549 cellliquid chromatography-mass spectrometryuntargeted analysisHomo sapienstargeted metabolite profilingU-13C glucose tracingexperimental sampleLC-MS analysis was performed with an Orbitrap Eclipse Tribrid mass spectrometerer (ThermoFisher Scientific, San Jose, CA) run with polarity switching (+3.4 kV/- 2.6 kV) in full scan mode with an m/z range of 65-900 and 60k resolution.false13C-glucose tracing analysis of IFN-treated A549 cells expressing wild-type SARS-CoV-2 Nsp3-4 or a deISGylation-defective Nsp3-4 mutant proteinInterferon (IFN)-stimulated gene 15 (ISG15) regulates diverse cellular processes, including antiviral immunity, through its conjugation to target proteins (ISGylation). Increasing evidence suggests that ISGylation can also reshape cellular metabolism; however, how viruses counteract ISGylation-mediated metabolic rewiring remains poorly understood. This analysis investigated the impact of the deISGylation activity of the SARS-CoV-2 papain-like protease, which is part of the Nsp3 protein, on type I IFN-driven modulation of cellular glucose metabolism. A549 cells expressing empty vector, wild-type (WT) Nsp3-4 from SARS-CoV-2, or a mutant Nsp3-4 protein that is deficient in PLpro deISGylation activity, were treated with IFNα (or mock-treated) and then subjected to 13C-glucose tracing analysis. Following isotope labeling, intracellular metabolites were extracted and analyzed by LC-MS to assess 13C incorporation into intermediates of glycolysis and the pentose phosphate pathway (PPP). Comparative analysis of the results revealed distinct labeling patterns in glycolytic and PPP metabolites under IFN-stimulated conditions, supporting a role for PLpro deISGylation activity in modulating cellular glucose metabolism. These data provide insight into how SARS-CoV-2 Nsp3 counteracts IFN-induced metabolic rewiring through its deISGylation activity.2026-05-052026-03-11MTBLS14030