#METABOLOMICS WORKBENCH estancliffe_20210128_125700 DATATRACK_ID:2432 STUDY_ID:ST001853 ANALYSIS_ID:AN003004 PROJECT_ID:000000 VERSION 1 CREATED_ON June 29, 2021, 1:55 pm #PROJECT PR:PROJECT_TITLE Longitudinal Metabolomics of Human Plasma Reveals Robust Prognostic Markers of PR:PROJECT_TITLE COVID-19 Disease Severity PR:PROJECT_SUMMARY There is an urgent need to identify which COVID-19 patients will develop PR:PROJECT_SUMMARY life-threatening illness so that medical resources can be optimally allocated PR:PROJECT_SUMMARY and rapid treatment can be administered early in the disease course, when PR:PROJECT_SUMMARY clinical management is most effective. To aid in the prognostic classification PR:PROJECT_SUMMARY of disease severity, we perform untargeted metabolomics on plasma from 339 PR:PROJECT_SUMMARY patients, with samples collected at six longitudinal time points. Using the PR:PROJECT_SUMMARY temporal metabolic profiles and machine learning, we build a predictive model of PR:PROJECT_SUMMARY disease severity. We discover that a panel of metabolites measured at the time PR:PROJECT_SUMMARY of study entry successfully determine disease severity. Through analysis of PR:PROJECT_SUMMARY longitudinal samples, we confirm that the majority of these markers are directly PR:PROJECT_SUMMARY related to disease progression and that their levels are restored to baseline PR:PROJECT_SUMMARY upon disease recovery. Finally, we validate that these metabolites are also PR:PROJECT_SUMMARY altered in a hamster model of COVID-19. Our results indicate that metabolic PR:PROJECT_SUMMARY changes associated with COVID-19 severity can be effectively used to stratify PR:PROJECT_SUMMARY patients and inform resource allocation during the pandemic. PR:INSTITUTE Washington University, St. Louis PR:DEPARTMENT Chemistry PR:LABORATORY Patti PR:LAST_NAME Patti PR:FIRST_NAME Gary PR:ADDRESS McMillen Chemistry Laboratory, Washington University 1 Brookings Dr @ Throop PR:ADDRESS Drive, Rm 102, St. Louis, MO 63130-4899 PR:EMAIL gjpattij@wustl.edu PR:PHONE 314-935-3512 #STUDY ST:STUDY_TITLE Longitudinal Metabolomics of Human Plasma Reveals Robust Prognostic Markers of ST:STUDY_TITLE COVID-19 Disease Severity ST:STUDY_SUMMARY There is an urgent need to identify which COVID-19 patients will develop ST:STUDY_SUMMARY life-threatening illness so that medical resources can be optimally allocated ST:STUDY_SUMMARY and rapid treatment can be administered early in the disease course, when ST:STUDY_SUMMARY clinical management is most effective. To aid in the prognostic classification ST:STUDY_SUMMARY of disease severity, we perform untargeted metabolomics on plasma from 339 ST:STUDY_SUMMARY patients, with samples collected at six longitudinal time points. Using the ST:STUDY_SUMMARY temporal metabolic profiles and machine learning, we build a predictive model of ST:STUDY_SUMMARY disease severity. We discover that a panel of metabolites measured at the time ST:STUDY_SUMMARY of study entry successfully determine disease severity. Through analysis of ST:STUDY_SUMMARY longitudinal samples, we confirm that the majority of these markers are directly ST:STUDY_SUMMARY related to disease progression and that their levels are restored to baseline ST:STUDY_SUMMARY upon disease recovery. Finally, we validate that these metabolites are also ST:STUDY_SUMMARY altered in a hamster model of COVID-19. Our results indicate that metabolic ST:STUDY_SUMMARY changes associated with COVID-19 severity can be effectively used to stratify ST:STUDY_SUMMARY patients and inform resource allocation during the pandemic. ST:INSTITUTE Washington University, St. Louis ST:DEPARTMENT Chemistry ST:LABORATORY Patti ST:LAST_NAME Patti ST:FIRST_NAME Gary ST:ADDRESS McMillen Chemistry Laboratory, Washington University 1 Brookings Dr @ Throop ST:ADDRESS Drive, Rm 102, St. Louis, MO 63130-4899 ST:EMAIL gjpattij@wustl.edu ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 56 ST:NUM_FEMALES 56 ST:PHONE 314-935-3512 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mesocricetus auratus #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - P H-433 Group:Influenza | Day post infection:4 RAW_FILE_NAME=P H-433_polar_pos.mzML,P H-433_polar_neg.mzML,P H-433_lipid_pos.mzML,P H-433_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-457 Group:Influenza | Day post infection:4 RAW_FILE_NAME=P H-457_polar_pos.mzML,P H-457_polar_neg.mzML,P H-457_lipid_pos.mzML,P H-457_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-458 Group:Influenza | Day post infection:4 RAW_FILE_NAME=P H-458_polar_pos.mzML,P H-458_polar_neg.mzML,P H-458_lipid_pos.mzML,P H-458_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-459 Group:SARS-CoV-2 | Day post infection:14 RAW_FILE_NAME=P H-459_polar_pos.mzML,P H-459_polar_neg.mzML,P H-459_lipid_pos.mzML,P H-459_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-460 Group:Influenza | Day post infection:4 RAW_FILE_NAME=P H-460_polar_pos.mzML,P H-460_polar_neg.mzML,P H-460_lipid_pos.mzML,P H-460_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-461 Group:Influenza | Day post infection:6 RAW_FILE_NAME=P H-461_polar_pos.mzML,P H-461_polar_neg.mzML,P H-461_lipid_pos.mzML,P H-461_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-462 Group:Influenza | Day post infection:6 RAW_FILE_NAME=P H-462_polar_pos.mzML,P H-462_polar_neg.mzML,P H-462_lipid_pos.mzML,P H-462_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-463 Group:SARS-CoV-2 | Day post infection:6 RAW_FILE_NAME=P H-463_polar_pos.mzML,P H-463_polar_neg.mzML,P H-463_lipid_pos.mzML,P H-463_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-464 Group:Mock | Day post infection:4 RAW_FILE_NAME=P H-464_polar_pos.mzML,P H-464_polar_neg.mzML,P H-464_lipid_pos.mzML,P H-464_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-466 Group:Influenza | Day post infection:4 RAW_FILE_NAME=P H-466_polar_pos.mzML,P H-466_polar_neg.mzML,P H-466_lipid_pos.mzML,P H-466_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-467 Group:Influenza | Day post infection:14 RAW_FILE_NAME=P H-467_polar_pos.mzML,P H-467_polar_neg.mzML,P H-467_lipid_pos.mzML,P H-467_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-468 Group:SARS-CoV-2 | Day post infection:6 RAW_FILE_NAME=P H-468_polar_pos.mzML,P H-468_polar_neg.mzML,P H-468_lipid_pos.mzML,P H-468_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-469 -1 Group:SARS-CoV-2 | Day post infection:2 RAW_FILE_NAME=P H-469 -1_polar_pos.mzML,P H-469 -1_polar_neg.mzML,P H-469 -1_lipid_pos.mzML,P H-469 -1_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-469 -2 Group:SARS-CoV-2 | Day post infection:4 RAW_FILE_NAME=P H-469 -2 _polar_pos.mzML,P H-469 -2 _polar_neg.mzML,P H-469 -2 _lipid_pos.mzML,P H-469 -2 _lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-470 Group:Mock | Day post infection:14 RAW_FILE_NAME=P H-470_polar_pos.mzML,P H-470_polar_neg.mzML,P H-470_lipid_pos.mzML,P H-470_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-471 Group:SARS-CoV-2 | Day post infection:4 RAW_FILE_NAME=P H-471_polar_pos.mzML,P H-471_polar_neg.mzML,P H-471_lipid_pos.mzML,P H-471_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-472 Group:Mock | Day post infection:4 RAW_FILE_NAME=P H-472_polar_pos.mzML,P H-472_polar_neg.mzML,P H-472_lipid_pos.mzML,P H-472_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-473 Group:Influenza | Day post infection:14 RAW_FILE_NAME=P H-473_polar_pos.mzML,P H-473_polar_neg.mzML,P H-473_lipid_pos.mzML,P H-473_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-474 Group:Influenza | Day post infection:2 RAW_FILE_NAME=P H-474_polar_pos.mzML,P H-474_polar_neg.mzML,P H-474_lipid_pos.mzML,P H-474_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-475 Group:Mock | Day post infection:14 RAW_FILE_NAME=P H-475_polar_pos.mzML,P H-475_polar_neg.mzML,P H-475_lipid_pos.mzML,P H-475_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-476 Group:Mock | Day post infection:4 RAW_FILE_NAME=P H-476_polar_pos.mzML,P H-476_polar_neg.mzML,P H-476_lipid_pos.mzML,P H-476_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-477 Group:Influenza | Day post infection:6 RAW_FILE_NAME=P H-477_polar_pos.mzML,P H-477_polar_neg.mzML,P H-477_lipid_pos.mzML,P H-477_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-478 Group:Influenza | Day post infection:2 RAW_FILE_NAME=P H-478_polar_pos.mzML,P H-478_polar_neg.mzML,P H-478_lipid_pos.mzML,P H-478_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-479 Group:SARS-CoV-2 | Day post infection:14 RAW_FILE_NAME=P H-479_polar_pos.mzML,P H-479_polar_neg.mzML,P H-479_lipid_pos.mzML,P H-479_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-480 Group:Mock | Day post infection:4 RAW_FILE_NAME=P H-480_polar_pos.mzML,P H-480_polar_neg.mzML,P H-480_lipid_pos.mzML,P H-480_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-481 Group:Mock | Day post infection:14 RAW_FILE_NAME=P H-481_polar_pos.mzML,P H-481_polar_neg.mzML,P H-481_lipid_pos.mzML,P H-481_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-482 Group:Mock | Day post infection:4 RAW_FILE_NAME=P H-482_polar_pos.mzML,P H-482_polar_neg.mzML,P H-482_lipid_pos.mzML,P H-482_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-483 Group:Influenza | Day post infection:14 RAW_FILE_NAME=P H-483_polar_pos.mzML,P H-483_polar_neg.mzML,P H-483_lipid_pos.mzML,P H-483_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-484 Group:SARS-CoV-2 | Day post infection:14 RAW_FILE_NAME=P H-484_polar_pos.mzML,P H-484_polar_neg.mzML,P H-484_lipid_pos.mzML,P H-484_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-485 Group:SARS-CoV-2 | Day post infection:14 RAW_FILE_NAME=P H-485_polar_pos.mzML,P H-485_polar_neg.mzML,P H-485_lipid_pos.mzML,P H-485_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-486 Group:Influenza | Day post infection:6 RAW_FILE_NAME=P H-486_polar_pos.mzML,P H-486_polar_neg.mzML,P H-486_lipid_pos.mzML,P H-486_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-487 Group:SARS-CoV-2 | Day post infection:4 RAW_FILE_NAME=P H-487_polar_pos.mzML,P H-487_polar_neg.mzML,P H-487_lipid_pos.mzML,P H-487_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-488 Group:SARS-CoV-2 | Day post infection:4 RAW_FILE_NAME=P H-488_polar_pos.mzML,P H-488_polar_neg.mzML,P H-488_lipid_pos.mzML,P H-488_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-489 Group:Mock | Day post infection:14 RAW_FILE_NAME=P H-489_polar_pos.mzML,P H-489_polar_neg.mzML,P H-489_lipid_pos.mzML,P H-489_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-490 Group:Mock | Day post infection:14 RAW_FILE_NAME=P H-490_polar_pos.mzML,P H-490_polar_neg.mzML,P H-490_lipid_pos.mzML,P H-490_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-491 Group:Influenza | Day post infection:6 RAW_FILE_NAME=P H-491_polar_pos.mzML,P H-491_polar_neg.mzML,P H-491_lipid_pos.mzML,P H-491_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-492 Group:Influenza | Day post infection:2 RAW_FILE_NAME=P H-492_polar_pos.mzML,P H-492_polar_neg.mzML,P H-492_lipid_pos.mzML,P H-492_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-495 Group:SARS-CoV-2 | Day post infection:6 RAW_FILE_NAME=P H-495_polar_pos.mzML,P H-495_polar_neg.mzML,P H-495_lipid_pos.mzML,P H-495_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-496 Group:Influenza | Day post infection:14 RAW_FILE_NAME=P H-496_polar_pos.mzML,P H-496_polar_neg.mzML,P H-496_lipid_pos.mzML,P H-496_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-497 Group:Influenza | Day post infection:14 RAW_FILE_NAME=P H-497_polar_pos.mzML,P H-497_polar_neg.mzML,P H-497_lipid_pos.mzML,P H-497_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-498 Group:Influenza | Day post infection:4 RAW_FILE_NAME=P H-498_polar_pos.mzML,P H-498_polar_neg.mzML,P H-498_lipid_pos.mzML,P H-498_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-499 Group:Influenza | Day post infection:14 RAW_FILE_NAME=P H-499_polar_pos.mzML,P H-499_polar_neg.mzML,P H-499_lipid_pos.mzML,P H-499_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-501 Group:Mock | Day post infection:14 RAW_FILE_NAME=P H-501_polar_pos.mzML,P H-501_polar_neg.mzML,P H-501_lipid_pos.mzML,P H-501_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-502 Group:Influenza | Day post infection:2 RAW_FILE_NAME=P H-502_polar_pos.mzML,P H-502_polar_neg.mzML,P H-502_lipid_pos.mzML,P H-502_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-504 Group:SARS-CoV-2 | Day post infection:6 RAW_FILE_NAME=P H-504_polar_pos.mzML,P H-504_polar_neg.mzML,P H-504_lipid_pos.mzML,P H-504_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-505 Group:Influenza | Day post infection:2 RAW_FILE_NAME=P H-505_polar_pos.mzML,P H-505_polar_neg.mzML,P H-505_lipid_pos.mzML,P H-505_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-506 Group:Influenza | Day post infection:2 RAW_FILE_NAME=P H-506_polar_pos.mzML,P H-506_polar_neg.mzML,P H-506_lipid_pos.mzML,P H-506_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-507 Group:Mock | Day post infection:4 RAW_FILE_NAME=P H-507_polar_pos.mzML,P H-507_polar_neg.mzML,P H-507_lipid_pos.mzML,P H-507_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-509 Group:SARS-CoV-2 | Day post infection:6 RAW_FILE_NAME=P H-509_polar_pos.mzML,P H-509_polar_neg.mzML,P H-509_lipid_pos.mzML,P H-509_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-510 Group:SARS-CoV-2 | Day post infection:14 RAW_FILE_NAME=P H-510_polar_pos.mzML,P H-510_polar_neg.mzML,P H-510_lipid_pos.mzML,P H-510_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-511 Group:SARS-CoV-2 | Day post infection:14 RAW_FILE_NAME=P H-511_polar_pos.mzML,P H-511_polar_neg.mzML,P H-511_lipid_pos.mzML,P H-511_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-512 Group:SARS-CoV-2 | Day post infection:6 RAW_FILE_NAME=P H-512_polar_pos.mzML,P H-512_polar_neg.mzML,P H-512_lipid_pos.mzML,P H-512_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-513 Group:SARS-CoV-2 | Day post infection:2 RAW_FILE_NAME=P H-513_polar_pos.mzML,P H-513_polar_neg.mzML,P H-513_lipid_pos.mzML,P H-513_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-514 Group:SARS-CoV-2 | Day post infection:4 RAW_FILE_NAME=P H-514_polar_pos.mzML,P H-514_polar_neg.mzML,P H-514_lipid_pos.mzML,P H-514_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-515 Group:Influenza | Day post infection:6 RAW_FILE_NAME=P H-515_polar_pos.mzML,P H-515_polar_neg.mzML,P H-515_lipid_pos.mzML,P H-515_lipid_neg.mzML SUBJECT_SAMPLE_FACTORS - P H-516 Group:SARS-CoV-2 | Day post infection:2 RAW_FILE_NAME=P H-516_polar_pos.mzML,P H-516_polar_neg.mzML,P H-516_lipid_pos.mzML,P H-516_lipid_neg.mzML #COLLECTION CO:COLLECTION_SUMMARY Outbred female LVG golden Syrian hamsters (6-8 weeks of age) were obtained from CO:COLLECTION_SUMMARY Charles River Laboratories (Kingston, NY). The hamsters were anesthetized by CO:COLLECTION_SUMMARY intraperitoneal injection of a mixture of ketamine and xylazine prior to CO:COLLECTION_SUMMARY intranasal inoculation with 0.1 mL of 1e5 plaque-forming units (PFU) of CO:COLLECTION_SUMMARY SARS-CoV-2 (WA-1) or H1N1 influenza A virus (A/California/04/2009). On day 2, 4, CO:COLLECTION_SUMMARY 6, and 14 after infection, 3-6 anesthetized hamsters per infection group were CO:COLLECTION_SUMMARY euthanized by exsanguination followed by intracardiac injection of veterinary CO:COLLECTION_SUMMARY euthanasia solution (SleepAway; Fort Dodge). Plasma samples were treated by CO:COLLECTION_SUMMARY exposure to germicidal UV-C light. CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY The hamsters were anesthetized by intraperitoneal injection of a mixture of TR:TREATMENT_SUMMARY ketamine and xylazine prior to intranasal inoculation with 0.1 mL of 1e5 TR:TREATMENT_SUMMARY plaque-forming units (PFU) of SARS-CoV-2 (WA-1) or H1N1 influenza A virus TR:TREATMENT_SUMMARY (A/California/04/2009). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Hamster plasma samples were diluted 1:4 with methanol (v/v), vortexed for 30 SP:SAMPLEPREP_SUMMARY seconds, and incubated at -20 C for 2 hours. Samples were centrifuged for 10 SP:SAMPLEPREP_SUMMARY minutes at 13,500 x g at 4°C and supernatant was transferred to a new SP:SAMPLEPREP_SUMMARY centrifuge tube, concentrated, and stored at -80 C until reconstitution. Hamster SP:SAMPLEPREP_SUMMARY plasma was thawed on ice. A 50 µL aliquot was transferred onto the SP:SAMPLEPREP_SUMMARY solid-phase-extraction (SPE)-system CAPTIVA-EMR Lipid 96-wellplate (Agilent SP:SAMPLEPREP_SUMMARY Technologies) before addition of 250 µL of acetonitrile containing 1% formic SP:SAMPLEPREP_SUMMARY acid (v/v) and 10 µM internal standard (consisting of uniformly 13C and 15N SP:SAMPLEPREP_SUMMARY labeled amino acids from Cambridge Isotope Laboratories, Inc). The samples were SP:SAMPLEPREP_SUMMARY mixed for 1 min at 360 rpm on an orbital shaker at room temperature prior to a SP:SAMPLEPREP_SUMMARY 10 min incubation period at 4 C. Afterwards, 200 µL 80% acetonitrile in SP:SAMPLEPREP_SUMMARY water (v/v) were added to the samples. The samples were mixed on an orbital SP:SAMPLEPREP_SUMMARY shaker (360 rpm) for an additional 10 min at room temperature. The samples were SP:SAMPLEPREP_SUMMARY then eluted into a 96-deepwell collection plate by centrifugation (10 min, 57 x SP:SAMPLEPREP_SUMMARY g, 4 C followed by 2 min, 1000 x g, 4 C). Polar eluates were stored at -80 C SP:SAMPLEPREP_SUMMARY until the day of LC/MS analysis. The SPE-plates were then washed twice with 500 SP:SAMPLEPREP_SUMMARY µL 80% acetonitrile in water (v/v). Lipids still bound to the SPE-material were SP:SAMPLEPREP_SUMMARY then released into a second elution plate, in two elution steps applying 2x 500 SP:SAMPLEPREP_SUMMARY µL 1:1 methyl tert-butyl ether:methanol (v/v) onto the SPE cartridge and SP:SAMPLEPREP_SUMMARY centrifuging for 2 min at 1000 g and 4 C. The combined eluates were dried under SP:SAMPLEPREP_SUMMARY a stream of nitrogen (Biotage SPE Dry Evaporation System) at room temperature SP:SAMPLEPREP_SUMMARY and reconstituted with 100 µL 1:1 2-propanol:methanol (v/v) prior to LC/MS SP:SAMPLEPREP_SUMMARY analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY An aliquot of 2 µL of lipid extract was subjected to LC/MS analysis by using an CH:CHROMATOGRAPHY_SUMMARY Agilent 1290 Infinity II LC-system coupled to an Agilent 6545 Q-TOF mass CH:CHROMATOGRAPHY_SUMMARY spectrometer with a dual Agilent Jet Stream electrospray ionization source. CH:CHROMATOGRAPHY_SUMMARY Lipids were separated on an Acquity UPLC® HSS T3 column (2.1 x 150 mm, 1.8 µm) CH:CHROMATOGRAPHY_SUMMARY including an Acquity UPLC® HSS T3 VanGuard Pre-Column (2.1 x 5mm, 1.8 µm) at a CH:CHROMATOGRAPHY_SUMMARY temperature of 60 C and a flow rate of 250 µLmin-1. The mobile phases CH:CHROMATOGRAPHY_SUMMARY consisted of A: 60% acetonitrile, 40% water, 0.1% formic acid, 10 mM ammonium CH:CHROMATOGRAPHY_SUMMARY formate, 2.5 µM medronic acid, and B: 90% 2-propanol, 10% acetonitrile, 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid, 10 mM ammonium formate (dissolved in 1 mL water). The following CH:CHROMATOGRAPHY_SUMMARY linear gradient was used: 0-2 min, 30% B; 17 min, 75% B; 20 min, 85%; 23-26 min, CH:CHROMATOGRAPHY_SUMMARY 100% B; 26, 30% B followed by a re-equilibration phase of 5 min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Acquity UPLC® HSS T3 column (2.1 x 150 mm, 1.8 µm) including an Acquity UPLC® CH:COLUMN_NAME HSS T3 VanGuard Pre-Column (2.1 x 5mm, 1.8 µm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS gas temperature 250 C, drying gas flow 11 L*min-1, nebulizer pressure 35 psi, MS:MS_COMMENTS sheath gas temperature 300 C, sheath gas flow 12 Lmin-1, VCap 3000 V, MS:MS_COMMENTS nozzle voltage 500 V, Fragmentor 160 V, Skimmer 65 V, Oct 1 RF Vpp 750 V, and MS:MS_COMMENTS m/z range 50-1700. Data were acquired under continuous reference mass correction MS:MS_COMMENTS at m/z 121.0509 and 922.0890 in positive ion mode and m/z 119.0363 and 966.0007 MS:MS_COMMENTS in negative ion mode. Polar metabolite identifications were supported by MS:MS_COMMENTS matching the retention time, accurate mass, and MS/MS fragmentation data to our MS:MS_COMMENTS in-house retention time and MS/MS library created from authentic reference MS:MS_COMMENTS standards (Mass Spectrometry Metabolite Library supplied by IROA Technologies, MS:MS_COMMENTS Millipore Sigma, St. Louis, MO, USA) and online MS/MS libraries (Human MS:MS_COMMENTS Metabolome Database (HMDB, https://hmdb.ca), Mass Bank of North America (MoNA, MS:MS_COMMENTS https://mona.fiehnlab.ucdavis.edu/), and mzCloud (https://mzcloud.org). Lipid MS:MS_COMMENTS iterative MS/MS data were annotated with the Agilent Lipid Annotator software. MS:MS_COMMENTS All data files were then analyzed in Skyline (Version 20.1.0.155) to obtain peak MS:MS_COMMENTS areas. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Relative Intensity MS_METABOLITE_DATA_START Samples P H-433 P H-457 P H-458 P H-459 P H-460 P H-461 P H-462 P H-463 P H-464 P H-466 P H-467 P H-468 P H-469 -1 P H-469 -2 P H-470 P H-471 P H-472 P H-473 P H-474 P H-475 P H-476 P H-477 P H-478 P H-479 P H-480 P H-481 P H-482 P H-483 P H-484 P H-485 P H-486 P H-487 P H-488 P H-489 P H-490 P H-491 P H-492 P H-495 P H-496 P H-497 P H-498 P H-499 P H-501 P H-502 P H-504 P H-505 P H-506 P H-507 P H-509 P H-510 P H-511 P H-512 P H-513 P H-514 P H-515 P H-516 Factors Group:Influenza | Day post infection:4 Group:Influenza | Day post infection:4 Group:Influenza | Day post infection:4 Group:SARS-CoV-2 | Day post infection:14 Group:Influenza | Day post infection:4 Group:Influenza | Day post infection:6 Group:Influenza | Day post infection:6 Group:SARS-CoV-2 | Day post infection:6 Group:Mock | Day post infection:4 Group:Influenza | Day post infection:4 Group:Influenza | Day post infection:14 Group:SARS-CoV-2 | Day post infection:6 Group:SARS-CoV-2 | Day post infection:2 Group:SARS-CoV-2 | Day post infection:4 Group:Mock | Day post infection:14 Group:SARS-CoV-2 | Day post infection:4 Group:Mock | Day post infection:4 Group:Influenza | Day post infection:14 Group:Influenza | Day post infection:2 Group:Mock | Day post infection:14 Group:Mock | Day post infection:4 Group:Influenza | Day post infection:6 Group:Influenza | Day post infection:2 Group:SARS-CoV-2 | Day post infection:14 Group:Mock | Day post infection:4 Group:Mock | Day post infection:14 Group:Mock | Day post infection:4 Group:Influenza | Day post infection:14 Group:SARS-CoV-2 | Day post infection:14 Group:SARS-CoV-2 | Day post infection:14 Group:Influenza | Day post infection:6 Group:SARS-CoV-2 | Day post infection:4 Group:SARS-CoV-2 | Day post infection:4 Group:Mock | Day post infection:14 Group:Mock | Day post infection:14 Group:Influenza | Day post infection:6 Group:Influenza | Day post infection:2 Group:SARS-CoV-2 | Day post infection:6 Group:Influenza | Day post infection:14 Group:Influenza | Day post infection:14 Group:Influenza | Day post infection:4 Group:Influenza | Day post infection:14 Group:Mock | Day post infection:14 Group:Influenza | Day post infection:2 Group:SARS-CoV-2 | Day post infection:6 Group:Influenza | Day post infection:2 Group:Influenza | Day post infection:2 Group:Mock | Day post infection:4 Group:SARS-CoV-2 | Day post infection:6 Group:SARS-CoV-2 | Day post infection:14 Group:SARS-CoV-2 | Day post infection:14 Group:SARS-CoV-2 | Day post infection:6 Group:SARS-CoV-2 | Day post infection:2 Group:SARS-CoV-2 | Day post infection:4 Group:Influenza | Day post infection:6 Group:SARS-CoV-2 | Day post infection:2 Cer_NS d18:2_16:0 68374 65549 61692 97078 52750 123486 100931 102544 70498 626204 55611 72375 194827 49265 83494 110670 299743 71989 209161 58616 254835 233257 113172 100782 415292 175207 87579 138990 57039 91384 288342 252931 115782 311563 76973 3905 237059 73758 116033 195411 73115 113357 68763 206502 167559 404090 132536 25281 194436 101028 72893 28425 130360 124234 112588 99824 PE 16:0_18:2 452595 287439 203791 973719 521200 770970 353824 773061 757250 373608 310846 742228 562208 710019 482139 1020264 1061455 704792 1065997 344578 371210 1038583 874134 1156423 676663 1011527 379839 674583 712173 381219 857317 220732 680996 1387477 453818 4805 958897 488504 786587 338232 310868 868044 643263 666904 664147 308608 573628 534865 450243 921397 538965 325012 968536 920223 878967 694608 PE 16:0_20:4 58843 31924 43207 110559 71229 72179 42209 102436 110001 44834 30237 91804 93777 119130 45673 129235 106268 90305 176930 46079 40360 111168 107893 149707 86312 102380 57093 49232 98023 48353 100656 134984 108039 125856 50422 89544 121642 73934 83754 52482 45329 108383 83040 85372 85638 59784 83235 57734 76565 113991 57839 51065 111927 107537 106450 96229 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Cer_NS d18:2_16:0 PE 16:0_18:2 PE 16:0_20:4 METABOLITES_END #END