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MB Sample ID: SA323834
| Local Sample ID: | 32 |
| Subject ID: | SU003097 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Male and female |
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Subject:
| Subject ID: | SU003097 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Male and female |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 32 | SA323834 | FL038670 | mild | Symotons |
Collection:
| Collection ID: | CO003090 |
| Collection Summary: | Residual serum samples for biochemical and immunological tests were collected from patients with suspected SARS-CoV-2 infection who underwent RT-PCR testing of nasopharyngeal swab or saliva samples at Keio University Hospital or Osaka Metropolitan University Hospital from March 2020 to January 2021. The diagnosis of COVID-19 was based on a positive RT-PCR test from a nasopharyngeal swab or saliva sample. The classification of severity was based on the Japanese Ministry of Health, Labour and Welfare’s classification of severity (criteria for evaluation by healthcare professionals) and was defined as follows: asymptomatic, no symptoms associated with COVID-19; mild disease, SARS-CoV-2-positive with no findings of pneumonia; moderate disease, SARS-CoV-2-positive with findings of pneumonia but not requiring a ventilator or intensive care unit (ICU) management; and severe disease, SARS-CoV-2-positive requiring ventilator or ICU management, including death attributed to COVID-19 during the treatment period. For a negative control, residual serum samples were collected from patients who underwent RT-PCR testing using nasopharyngeal swab fluid between April and May 2020 at Keio University Hospital and received negative RT-PCR test results, making them clinically unlikely to have COVID-19. The study included the early patient cohorts of waves 1, 2, and 3 of COVID-19 conducted in Japan from March to December 2020. For cohorts 1 and 2, patients were not excluded because of comorbidities, as the main objective was to include the greatest possible number of severe cases for which specimens were available early after disease onset. Cases in the mild and moderate groups were selected based on matching age and sex distribution with the severe group. No specific exclusions were made in any group during the study period, mainly because the number of samples was limited. Asymptomatic and mildly symptomatic patients were selected for inclusion by balancing the sex ratio. The higher proportion of males in the moderate and severe categories is due to the observed tendency for males to be more severely affected. This was particularly true for patients hospitalized during the study period. Sample collection and utilization were conducted under the approval of the Ethics Committee of the Keio University School of Medicine (approval numbers 20200059 and 20200063) and the Ethics Committee of Osaka Metropolitan University Graduate School of Medicine (approval number 2020-003). The use of residual samples was conducted on an opt-out basis based on the approval of the relevant ethics committees. All samples were stored at -80 °C. |
| Sample Type: | Blood (serum) |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003106 |
| Treatment Summary: | No Treatment |
Sample Preparation:
| Sampleprep ID: | SP003103 |
| Sampleprep Summary: | Frozen serum samples of Cohort-1 participants, together with internal standard compounds were sonicated in ice-cold methanol (500 μL), to which an equal volume of chloroform and 0.4 times the volume of ultrapure water (LC/MS grade, Wako Pure Chemical, Tokyo, Japan) were added. The suspension was centrifuged at 15,000 × g for 15 min at 4 °C. The aqueous phase was then filtered in an ultrafiltration tube (Ultrafree MC-PLHCC, Human Metabolome Technologies, Tsuruoka City, Japan), and the filtrate was concentrated using a vacuum concentrator (SpeedVac; Thermo Fisher Scientific, Waltham, MA, USA). The concentrated filtrate was dissolved in 50 μL ultrapure water and subjected to IC-HR-MS analysis. Methionine sulfone (L-Met) and 2-morpholinoethanesulfonic acid (MES) were used as internal standards for cationic and anionic metabolites, respectively. |
Combined analysis:
| Analysis ID | AN004903 | AN004904 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Ion exchange | Reversed phase |
| Chromatography system | Thermo Dionex ICS-5000+ | Shimadzu Nexera X2 |
| Column | Dionex IonPac AS11-HC (250 x 2mm, 4um) | Sigma-Aldrich Discovery HS F5-3 (150 x 2.1 mm, 3 um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Triple quadrupole |
| MS instrument name | Thermo Q Exactive Focus | LCMS-8060, Shimadzu Corporation |
| Ion Mode | UNSPECIFIED | UNSPECIFIED |
| Units | Peak area/Internal Standard | Peak area/Internal Standard |
Chromatography:
| Chromatography ID: | CH003698 |
| Chromatography Summary: | Metabolites were detected using an orbitrap-type MS instrument (Q-Exactive focus; Thermo Fisher Scientific) connected to a high-performance IC system (ICS-5000+, Thermo Fisher Scientific) that enabled highly selective and sensitive metabolite quantification owing to the IC separation and Fourier transfer MS principle. The IC instrument was equipped with an anion electrolytic suppressor (Dionex AERS 500; Thermo Fisher Scientific) to convert the potassium hydroxide gradient into pure water before the sample entered the MS instrument. Separation was performed using a Dionex IonPac AS11-HC 4 μm particle size column (Thermo Fisher Scientific). The IC flow rate was 0.25 mL/min, supplemented post-column with 0.18 mL/min makeup flow of MeOH. The potassium hydroxide gradient conditions for IC separation were as follows: from 1 mM to 100 mM (0–40 min) to 100 mM (40–50 min) and to 1 mM (50.1–60 min) at a column temperature of 30 °C. The mass spectrometer was operated in the ESI-positive and negative mode with polarity switching, for all detections. A full mass scan (m/z 70–900) was performed at a resolution of 70,000. The automatic gain control target was set at 3 × 106 ions, and the maximum ion injection time was 100 ms. The source ionization parameters were optimized with a spray voltage of 3 kV, and other parameters were as follows: transfer temperature, 320 °C; S-Lens level, 50; heater temperature, 300 °C; sheath gas, 36; and aux gas, 10. |
| Instrument Name: | Thermo Dionex ICS-5000+ |
| Column Name: | Dionex IonPac AS11-HC (250 x 2mm, 4um) |
| Column Temperature: | 30 °C |
| Flow Gradient: | From 1 mM to 100 mM (0–40 min) to 100 mM (40–50 min) and to 1 mM (50.1–60 min) |
| Flow Rate: | 0.25 mL/min, supplemented post-column with 0.18 mL/min makeup flow of MeOH |
| Solvent A: | The potassium hydroxide gradient conditions |
| Solvent B: | N/A |
| Chromatography Type: | Ion exchange |
| Chromatography ID: | CH003699 |
| Chromatography Summary: | Cationic metabolite concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In essence, we employed a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) ion source (LCMS-8060, Shimadzu Corporation, Kyoto, Japan) operated in both positive and negative-ESI and in multiple reaction monitoring (MRM) modes. Analyte separation was achieved on a Discovery HS F5-3 column (2.1 mm I.D. × 150 mm L, 3 μm particle size; Sigma-Aldrich, St. Louis, MO, USA) through a gradient elution with mobile phase A (0.1% formate) and mobile phase B (0.1% acetonitrile). The elution profile was as follows: 100:0 (0–5 min), 75:25 (5–11 min), 65:35 (11–15 min), 5:95 (15–20 min), and 100:0 (20–25 min), with a constant flow rate of 0.25 mL/min and a column oven set at 40 °C. |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Sigma-Aldrich Discovery HS F5-3 (150 x 2.1 mm, 3 um) |
| Column Temperature: | 40 °C |
| Flow Gradient: | 100:0 (0–5 min), 75:25 (5–11 min), 65:35 (11–15 min), 5:95 (15–20 min), and 100:0 (20–25 min) |
| Flow Rate: | 0.25 mL/min |
| Solvent A: | 0.1% formate |
| Solvent B: | acetonitrile with 0.1% formate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004646 |
| Analysis ID: | AN004903 |
| Instrument Name: | Thermo Q Exactive Focus |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | For IC-HR-MS data analysis, the Trace Finder compound identification and confirmation setup parameters included a molecular ion intensity threshold override of 10,000, S/N 5, and mass tolerance of 5 ppm. Isotope pattern analysis using a 90% fit threshold, 30% allowable relative intensity deviation, and 5 ppm mass deviation was also performed to ensure that the relative intensities of the M + 1 and/or M + 2 isotope peaks for each compound were consistent with the theoretical relative intensities. |
| Ion Mode: | UNSPECIFIED |
| MS ID: | MS004647 |
| Analysis ID: | AN004904 |
| Instrument Name: | LCMS-8060, Shimadzu Corporation |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | For LC-MS/MS analysis, chromatographic peaks obtained with compound-specific SRM channels were integrated and manually reviewed. For a single target compound, one or more confirmatory SRM channels were set (if available) to confirm peak compound identification. Chromatograms were acquired using Lab Solutions (ver. 5.113, Shimadzu). Peak areas were determined using Data browser software. |
| Ion Mode: | UNSPECIFIED |
