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MB Sample ID: SA186718

Local Sample ID:	QC_6
Subject ID:	SU002074
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU002074
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
QC_6	SA186718	FL023088	QC	Label

Collection:

Collection ID:	CO002067
Collection Summary:	Blood samples were collected in EDTA tubes and then plasma was obtained.
Sample Type:	Blood (plasma)

Treatment:

Treatment ID:	TR002086
Treatment Summary:	Cryopreserved plasma was processed for virus inactivation by adding 1500 µL of cold methanol:ethanol (MeOH:EtOH) in a 1:1 (v/v) proportion to 500 µL of plasma. Then, samples were vortex-mixed for 1 min, incubated on ice for 5 min and centrifuged at 16,000 x g for 20 min at 4 °C to precipitate and remove proteins. The clean upper layer or supernatant, which contained the metabolites of interest, was transferred to Eppendorf tubes and stored at -80 °C until analysis.

Treatment ID:

TR002086

Treatment Summary:

Cryopreserved plasma was processed for virus inactivation by adding 1500 µL of cold methanol:ethanol (MeOH:EtOH) in a 1:1 (v/v) proportion to 500 µL of plasma. Then, samples were vortex-mixed for 1 min, incubated on ice for 5 min and centrifuged at 16,000 x g for 20 min at 4 °C to precipitate and remove proteins. The clean upper layer or supernatant, which contained the metabolites of interest, was transferred to Eppendorf tubes and stored at -80 °C until analysis.

Sample Preparation:

Sampleprep ID:	SP002080
Sampleprep Summary:	Two hundred microliters of frozen supernatant were thawed on ice and evaporated to dryness using a SpeedVac Concentrator System (Thermo Fisher Scientific, Waltham, MA). Then, it was resuspended in 100 µL of 0.2 mM methionine sulfone (MetS) in 0.1 M formic acid. Samples were vortex-mixed for 1 min, transferred to a Millipore filter (30 kDa protein cutoff) and centrifuged for 40 min at 2000 xg at 4 °C. Finally, the ultrafiltrate was transferred to a CE-MS vial for analysis. Quality control samples (QC) were prepared by pooling equal volumes of plasma supernatant from each sample and were treated as previously described. Finally, blank solutions were also prepared with MeOH:EtOH (1:1, v/v).

Sampleprep ID:

SP002080

Sampleprep Summary:

Two hundred microliters of frozen supernatant were thawed on ice and evaporated to dryness using a SpeedVac Concentrator System (Thermo Fisher Scientific, Waltham, MA). Then, it was resuspended in 100 µL of 0.2 mM methionine sulfone (MetS) in 0.1 M formic acid. Samples were vortex-mixed for 1 min, transferred to a Millipore filter (30 kDa protein cutoff) and centrifuged for 40 min at 2000 xg at 4 °C. Finally, the ultrafiltrate was transferred to a CE-MS vial for analysis. Quality control samples (QC) were prepared by pooling equal volumes of plasma supernatant from each sample and were treated as previously described. Finally, blank solutions were also prepared with MeOH:EtOH (1:1, v/v).

Combined analysis:

Analysis ID	AN003250
Analysis type	MS
Chromatography type	Unspecified
Chromatography system	7100 capillary electrophoresis (CE)
Column	fused-silica capillary from Agilent Technologies (total length,100 cm; inner diameter,50um)
MS Type	ESI
MS instrument type	Other
MS instrument name	Agilent 6230 TOF
Ion Mode	POSITIVE
Units	Peak Area

Chromatography:

Chromatography ID:	CH002395
Chromatography Summary:	Metabolite separation was carried out using a previously developed method in a fused-silica capillary from Agilent Technologies (total length, 100 cm; inner diameter, 50 µm) 1. Before each analysis, the capillary was flushed with background electrolyte (BGE) (1 M formic acid in 10% MeOH) for 5 min at 950 mbar. Samples were injected over 50 s at 50 mbar, and to improve the analysis reproducibility, BGE was injected for 10 s at 100 mbar after each sample injection. The separation was performed with an internal pressure of 25 mbar and 30 kV voltage, and the current observed under these conditions was 30 µA. The total analytical run time was 35 min.
Instrument Name:	7100 capillary electrophoresis (CE)
Column Name:	fused-silica capillary from Agilent Technologies (total length,100 cm; inner diameter,50um)
Chromatography Type:	Unspecified

MS:

MS ID:	MS003023
Analysis ID:	AN003250
Instrument Name:	Agilent 6230 TOF
Instrument Type:	Other
MS Type:	ESI
MS Comments:	The mass spectra data were acquired in positive polarity with a full scan range from 70 - 1000 m/z at a rate of 1.36 scan/s. The other MS parameters were as follows: fragmentor set to 125 V, skimmer to 65 V, OCT RF Vpp to 750 V, drying gas temperature to 200 °C, flow rate to 10 L/min, nebulizer to 10 psig, and capillary voltage to 3500 V. The sheath liquid contained two reference masses (5 µL of purine with m/z 121.0509 and 5 µL of HP-0921 with m/z 922.0098) in MeOH:H2O (1/1, v/v) with 1 mM formic acid, and the flow rate was 0.6 mL/min (1:100 split). The MS data were acquired using the Agilent MassHunter Workstation (version B.09.00, Agilent Technologies), and the raw data were inspected with the MassHunter Qualitative software (version B.08.00, Agilent Technologies) before data processing. CE-MS raw data were checked using MassHunter Qualitative software (version 10.0) to determine the data quality, the system mass accuracy and the reproducibility of QC injection and MetS area. Then, raw data were processed with MassHunter Profinder software (version 10.0 SP1) applying molecular feature extraction (MFE) to clean the data background and unrelated ions. MFE was also applied to find coeluting adducts (+H+ and +Na+ in positive ionization and neutral loss of water). Afterwards, the batch recursive feature extraction (RFE) algorithm, also included in the software, was used to align all the features across the samples by using the mass and retention time (RT). RFE re-extracts the batch files and improves the final compound group list, as it uses the previous results (mass and RT) obtained by the MFE algorithm to perform a more targeted extraction.
Ion Mode:	POSITIVE