#METABOLOMICS WORKBENCH OAD2021_20211116_073220 DATATRACK_ID:2931 STUDY_ID:ST001993 ANALYSIS_ID:AN003250 PROJECT_ID:PR001266 VERSION 1 CREATED_ON November 16, 2021, 8:40 am #PROJECT PR:PROJECT_TITLE Metabolic snapshot of plasma samples to study SARS-CoV-2 pathogenesis PR:PROJECT_SUMMARY MS based metabolomics was used in this study to analyze COVID-19 disease and the PR:PROJECT_SUMMARY susceptibility to SARS-CoV-2. In total, 63 plasma samples were analyzed and PR:PROJECT_SUMMARY different comparisons were performed. PR:INSTITUTE Universidad CEU San Pablo PR:DEPARTMENT Pharmacy PR:LABORATORY Centro de Metabolomica y Bioanálisis (CEMBIO) PR:LAST_NAME Barbas PR:FIRST_NAME Coral PR:ADDRESS CEU Universities, Urbanización Montepríncipe PR:EMAIL cbarbas@ceu.es PR:PHONE +34 913724700 #STUDY ST:STUDY_TITLE CE-MS based metabolomics to study plasma samples that reveal new pathways ST:STUDY_TITLE implicated in SARS-CoV-2 pathogenesis ST:STUDY_SUMMARY CE-MS based metabolomics was used in this study to analize COVID-19 disease and ST:STUDY_SUMMARY the susceptibility to SARS-CoV-2. In total, 63 plasma samples were analyzed and ST:STUDY_SUMMARY different comparisons were performed. To our knowledge, CE-MS has never been ST:STUDY_SUMMARY used to study COVID-19 and it is considered in this study an appropriate ST:STUDY_SUMMARY approach to extend the polar metabolome beyond what has been obtained by LC-MS ST:STUDY_SUMMARY and GC-MS ST:INSTITUTE Universidad CEU San Pablo ST:DEPARTMENT Pharmacy ST:LABORATORY Centro de Metabolomica y Bioanálisis (CEMBIO) ST:LAST_NAME Barbas ST:FIRST_NAME Coral ST:ADDRESS CEU Universities, Urbanización Montepríncipe ST:EMAIL cbarbas@ceu.es ST:PHONE +34 913724700 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - 2 Label:Healthy Gender=Male; Age=84; HTA=Yes; Obesity=No; CardiacDisease=Yes SUBJECT_SAMPLE_FACTORS - 7 Label:Mild_d0 Gender=Female; Age=55; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 8 Label:Mild_d0 Gender=Male; Age=59; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 9 Label:Mild_d0 Gender=Male; Age=49; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 12 Label:Moderate_d0 Gender=Male; Age=85; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 14 Label:Resistant Gender=Female; Age=30; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 15 Label:Resistant Gender=Female; Age=53; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 18 Label:Resistant Gender=Female; Age=51; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 21 Label:Resistant Gender=Female; Age=58; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 22 Label:Healthy Gender=Female; Age=38; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 23 Label:Healthy Gender=Female; Age=38; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 24 Label:Resistant Gender=Female; Age=34; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 25 Label:Resistant Gender=Male; Age=60; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 29 Label:Healthy Gender=Male; Age=56; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 31 Label:Healthy Gender=Male; Age=24; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 37 Label:Severe_d0 Gender=Female; Age=92; HTA=Yes; Obesity=No; CardiacDisease=Yes SUBJECT_SAMPLE_FACTORS - 43 Label:Resistant Gender=Female; Age=48; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 44 Label:Resistant Gender=Female; Age=48; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 45 Label:Resistant Gender=Female; Age=51; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 46 Label:Resistant Gender=Female; Age=42; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 47 Label:Resistant Gender=Male; Age=35; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 50 Label:Resistant Gender=Male; Age=29; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 51 Label:Resistant Gender=Female; Age=38; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 52 Label:Resistant Gender=Female; Age=28; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 53 Label:Resistant Gender=Female; Age=40; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 54 Label:Resistant Gender=Female; Age=44; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 55 Label:Resistant Gender=Male; Age=55; HTA=Yes; Obesity=Yes; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 56 Label:Resistant Gender=Female; Age=28; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 57 Label:Resistant Gender=Female; Age=46; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 58 Label:Resistant Gender=Male; Age=39; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 59 Label:Healthy Gender=Male; Age=36; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 60 Label:Resistant Gender=Female; Age=37; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 62 Label:Resistant Gender=Female; Age=36; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 63 Label:Resistant Gender=Female; Age=49; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 64 Label:Resistant Gender=Female; Age=48; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 65 Label:Resistant Gender=Female; Age=44; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 66 Label:Resistant Gender=Female; Age=57; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 70 Label:Resistant Gender=Female; Age=34; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 71 Label:Resistant Gender=Female; Age=38; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 72 Label:Resistant Gender=Female; Age=58; HTA=No; Obesity=Yes; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 1_0 Label:Severe_d0 Gender=Female; Age=59; HTA=No; Obesity=Yes; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 1_8 Label:Severe_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 10_0 Label:Moderate_d0 Gender=Male; Age=85; HTA=Yes; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 10_8 Label:Moderate_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 11_0 Label:Mild_d0 Gender=Female; Age=75; HTA=Yes; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 11_8 Label:Mild_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 12_8 Label:Moderate_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 17_0 Label:Mild_d0 Gender=Male; Age=57; HTA=Yes; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 17_8 Label:Mild_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 19_0 Label:Mild_d0 Gender=Female; Age=42; HTA=No; Obesity=Yes; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 19_8 Label:Mild_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 20_0 Label:Severe_d0 Gender=Male; Age=92; HTA=Yes; Obesity=No; CardiacDisease=Yes SUBJECT_SAMPLE_FACTORS - 20_8 Label:Severe_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 26_0 Label:Moderate_d0 Gender=Female; Age=91; HTA=Yes; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 26_8 Label:Moderate_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 3_0 Label:Moderate_d0 Gender=Male; Age=69; HTA=Yes; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 3_8 Label:Moderate_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 30_0 Label:Mild_d0 Gender=Male; Age=55; HTA=Yes; Obesity=Yes; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 30_8 Label:Mild_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 32_0 Label:Moderate_d0 Gender=Female; Age=92; HTA=Yes; Obesity=Yes; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 32_8 Label:Moderate_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 33_0 Label:Moderate_d0 Gender=Female; Age=41; HTA=Yes; Obesity=No; CardiacDisease=Yes SUBJECT_SAMPLE_FACTORS - 33_8 Label:Moderate_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 34_0 Label:Mild_d0 Gender=Male; Age=60; HTA=Yes; Obesity=Yes; CardiacDisease=Yes SUBJECT_SAMPLE_FACTORS - 34_8 Label:Mild_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 35_0 Label:Severe_d0 Gender=Female; Age=60; HTA=Yes; Obesity=Yes; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 35_8 Label:Severe_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 4_0 Label:Moderate_d0 Gender=Male; Age=76; HTA=Yes; Obesity=Yes; CardiacDisease=Yes SUBJECT_SAMPLE_FACTORS - 4_8 Label:Moderate_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 40_0 Label:Moderate_d0 Gender=Female; Age=85; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 40_8 Label:Moderate_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 41_0 Label:Severe_d0 Gender=Male; Age=51; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 41_8 Label:Severe_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 42_0 Label:Mild_d0 Gender=Female; Age=66; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 42_8 Label:Mild_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 48_0 Label:Mild_d0 Gender=Male; Age=50; HTA=Yes; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 48_8 Label:Mild_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 5_0 Label:Moderate_d0 Gender=Female; Age=89; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 5_8 Label:Moderate_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 67_0 Label:Mild_d0 Gender=Female; Age=84; HTA=Yes; Obesity=No; CardiacDisease=Yes SUBJECT_SAMPLE_FACTORS - 68_0 Label:Moderate_d0 Gender=Female; Age=75; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 69B Label:Resistant Gender=Male; Age=41; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 7_8 Label:Mild_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - 74_0 Label:Moderate_d0 Gender=Male; Age=78; HTA=No; Obesity=No; CardiacDisease=No SUBJECT_SAMPLE_FACTORS - 74_8 Label:Moderate_d8 Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_1 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_10 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_11 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_12 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_13 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_14 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_15 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_2 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_3 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_4 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_5 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_6 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_7 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_8 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- SUBJECT_SAMPLE_FACTORS - QC_9 Label:QC Gender=-; Age=-; HTA=-; Obesity=-; CardiacDisease=- #COLLECTION CO:COLLECTION_SUMMARY Blood samples were collected in EDTA tubes and then plasma was obtained. CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY Cryopreserved plasma was processed for virus inactivation by adding 1500 µL of TR:TREATMENT_SUMMARY cold methanol:ethanol (MeOH:EtOH) in a 1:1 (v/v) proportion to 500 µL of TR:TREATMENT_SUMMARY plasma. Then, samples were vortex-mixed for 1 min, incubated on ice for 5 min TR:TREATMENT_SUMMARY and centrifuged at 16,000 x g for 20 min at 4 °C to precipitate and remove TR:TREATMENT_SUMMARY proteins. The clean upper layer or supernatant, which contained the metabolites TR:TREATMENT_SUMMARY of interest, was transferred to Eppendorf tubes and stored at -80 °C until TR:TREATMENT_SUMMARY analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Two hundred microliters of frozen supernatant were thawed on ice and evaporated SP:SAMPLEPREP_SUMMARY to dryness using a SpeedVac Concentrator System (Thermo Fisher Scientific, SP:SAMPLEPREP_SUMMARY Waltham, MA). Then, it was resuspended in 100 µL of 0.2 mM methionine sulfone SP:SAMPLEPREP_SUMMARY (MetS) in 0.1 M formic acid. Samples were vortex-mixed for 1 min, transferred to SP:SAMPLEPREP_SUMMARY a Millipore filter (30 kDa protein cutoff) and centrifuged for 40 min at 2000 xg SP:SAMPLEPREP_SUMMARY at 4 °C. Finally, the ultrafiltrate was transferred to a CE-MS vial for SP:SAMPLEPREP_SUMMARY analysis. Quality control samples (QC) were prepared by pooling equal volumes of SP:SAMPLEPREP_SUMMARY plasma supernatant from each sample and were treated as previously described. SP:SAMPLEPREP_SUMMARY Finally, blank solutions were also prepared with MeOH:EtOH (1:1, v/v). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Metabolite separation was carried out using a previously developed method in a CH:CHROMATOGRAPHY_SUMMARY fused-silica capillary from Agilent Technologies (total length, 100 cm; inner CH:CHROMATOGRAPHY_SUMMARY diameter, 50 µm) 1. Before each analysis, the capillary was flushed with CH:CHROMATOGRAPHY_SUMMARY background electrolyte (BGE) (1 M formic acid in 10% MeOH) for 5 min at 950 CH:CHROMATOGRAPHY_SUMMARY mbar. Samples were injected over 50 s at 50 mbar, and to improve the analysis CH:CHROMATOGRAPHY_SUMMARY reproducibility, BGE was injected for 10 s at 100 mbar after each sample CH:CHROMATOGRAPHY_SUMMARY injection. The separation was performed with an internal pressure of 25 mbar and CH:CHROMATOGRAPHY_SUMMARY 30 kV voltage, and the current observed under these conditions was 30 µA. The CH:CHROMATOGRAPHY_SUMMARY total analytical run time was 35 min. CH:CHROMATOGRAPHY_TYPE Other CH:INSTRUMENT_NAME 7100 capillary electrophoresis (CE) CH:COLUMN_NAME fused-silica capillary from Agilent Technologies (total length, 100 cm; inner CH:COLUMN_NAME diameter, 50 µm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6230 TOF MS:INSTRUMENT_TYPE Other MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The mass spectra data were acquired in positive polarity with a full scan range MS:MS_COMMENTS from 70 - 1000 m/z at a rate of 1.36 scan/s. The other MS parameters were as MS:MS_COMMENTS follows: fragmentor set to 125 V, skimmer to 65 V, OCT RF Vpp to 750 V, drying MS:MS_COMMENTS gas temperature to 200 °C, flow rate to 10 L/min, nebulizer to 10 psig, and MS:MS_COMMENTS capillary voltage to 3500 V. The sheath liquid contained two reference masses (5 MS:MS_COMMENTS µL of purine with m/z 121.0509 and 5 µL of HP-0921 with m/z 922.0098) in MS:MS_COMMENTS MeOH:H2O (1/1, v/v) with 1 mM formic acid, and the flow rate was 0.6 mL/min MS:MS_COMMENTS (1:100 split). The MS data were acquired using the Agilent MassHunter MS:MS_COMMENTS Workstation (version B.09.00, Agilent Technologies), and the raw data were MS:MS_COMMENTS inspected with the MassHunter Qualitative software (version B.08.00, Agilent MS:MS_COMMENTS Technologies) before data processing. CE-MS raw data were checked using MS:MS_COMMENTS MassHunter Qualitative software (version 10.0) to determine the data quality, MS:MS_COMMENTS the system mass accuracy and the reproducibility of QC injection and MetS area. MS:MS_COMMENTS Then, raw data were processed with MassHunter Profinder software (version 10.0 MS:MS_COMMENTS SP1) applying molecular feature extraction (MFE) to clean the data background MS:MS_COMMENTS and unrelated ions. MFE was also applied to find coeluting adducts (+H+ and +Na+ MS:MS_COMMENTS in positive ionization and neutral loss of water). Afterwards, the batch MS:MS_COMMENTS recursive feature extraction (RFE) algorithm, also included in the software, was MS:MS_COMMENTS used to align all the features across the samples by using the mass and MS:MS_COMMENTS retention time (RT). RFE re-extracts the batch files and improves the final MS:MS_COMMENTS compound group list, as it uses the previous results (mass and RT) obtained by MS:MS_COMMENTS the MFE algorithm to perform a more targeted extraction. MS:MS_RESULTS_FILE ST001993_AN003250_Results.txt UNITS:Peak Area Has m/z:Yes Has RT:Yes RT units:Minutes #END