Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA242812

Local Sample ID:	C21
Subject ID:	SU002517
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	18-65
Gender:	Male and female
Human Exclusion Criteria:	HIV patients, respiratory infections beside TB, diabetes, chronic renal failure history and transplanted. individuals

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Subject:

Subject ID:	SU002517
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	18-65
Gender:	Male and female
Human Exclusion Criteria:	HIV patients, respiratory infections beside TB, diabetes, chronic renal failure history and transplanted. individuals

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
C21	SA242812	FL030402	Controls	Label

Collection:

Collection ID:	CO002510
Collection Summary:	Peripheral blood samples were collected at the Vendas Novas and Almada-Seixal Pneumonologic Diagnostic Centers (CDP-). Briefly, around 7 mL of blood was collected in order to obtain 3 mL of serum. Whole blood samples were harvested using Clot Activator Tubes (Monovette Serum Gel Z – 7.5 mL, S-monovette, Sarstedt®). IGRA test (QuantiFERON®-TB Gold IT, ©QIAGEN) was used to confirm infection status in Control and Latent groups. These samples were transported at 4°C to the National Institute of Health Doctor Ricardo Jorge (INSA). Samples displaying hemolysis or with unidentified IGRA results were excluded from further analysis.
Sample Type:	Blood (serum)

Treatment:

Treatment ID:	TR002529
Treatment Summary:	Blood was allowed to clot for three hours at 4°C after collection. The blood was then centrifuged at 1000 xg at 4°C for 30 min. Serum collected after centrifugation was passed through 0.2 μm filters (Sterile Acrodisc®, syringe filters with Supor membrane, 32 mm) to remove bacteria. Finally, an anti-protease cocktail (Protease Inhibitor Cocktail, ©SIGMA) was added to the filtered serum. The samples were transported from INSA to ITQB in a liquid nitrogen container and stored at −80°C.

Treatment ID:

TR002529

Treatment Summary:

Blood was allowed to clot for three hours at 4°C after collection. The blood was then centrifuged at 1000 xg at 4°C for 30 min. Serum collected after centrifugation was passed through 0.2 μm filters (Sterile Acrodisc®, syringe filters with Supor membrane, 32 mm) to remove bacteria. Finally, an anti-protease cocktail (Protease Inhibitor Cocktail, ©SIGMA) was added to the filtered serum. The samples were transported from INSA to ITQB in a liquid nitrogen container and stored at −80°C.

Sample Preparation:

Sampleprep ID:	SP002523
Sampleprep Summary:	Serum samples (100 µL) were extracted with 300 µL of methanol overnight at 4°C. The morning after, the samples were centrifuged for 5 min at 16.000 ×g and 4°C. The supernatant was transferred to a new tube, evaporated in the speedvac, and the pellet was stored at −20°C until further analysis. Previous to LC-MS/MS analysis the pellets were resuspended in 99.16 µL of H2O 0.1%FA. The Internal Standard (IS) – 3-Nitro-L-tyrosine – was added to a final concentration of 42 µM. In addition, four pools of samples were used for metabolite identification.

Sampleprep ID:

SP002523

Sampleprep Summary:

Serum samples (100 µL) were extracted with 300 µL of methanol overnight at 4°C. The morning after, the samples were centrifuged for 5 min at 16.000 ×g and 4°C. The supernatant was transferred to a new tube, evaporated in the speedvac, and the pellet was stored at −20°C until further analysis. Previous to LC-MS/MS analysis the pellets were resuspended in 99.16 µL of H2O 0.1%FA. The Internal Standard (IS) – 3-Nitro-L-tyrosine – was added to a final concentration of 42 µM. In addition, four pools of samples were used for metabolite identification.

Combined analysis:

Analysis ID	AN003951	AN003952
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Dionex UltiMate 3000 UHPLC	Dionex UltiMate 3000 UHPLC
Column	Waters XBridge C18 (50 x 2.1mm, 3um)	Waters XBridge C18 (50 x 2.1mm, 3um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Focus	Thermo Q Exactive Focus
Ion Mode	POSITIVE	NEGATIVE
Units	ua	au

Chromatography:

Chromatography ID:	CH002925
Instrument Name:	Dionex UltiMate 3000 UHPLC
Column Name:	Waters XBridge C18 (50 x 2.1mm, 3um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003686
Analysis ID:	AN003951
Instrument Name:	Thermo Q Exactive Focus
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Full-MS scan spectra were acquired in the m/z range 75 – 1125, at a resolution of 70,000 (full width at half maximum (FWHM) at m/z 200) and 1×106 automatic gain control (AGC). MS/MS scan spectra were acquired at 17,500 resolution (FWHM at m/z 200), with 1×105 AGC, maximum injection time of 100 ms and dynamic exclusion of 6 s. Raw data files were independently processed by Compound Discoverer™ 3.1 (ThermoFisher Scientific) software for metabolomics data analysis. The preferred database used for metabolite identification was mzCloud – since the "Search mzCloud '' node searches this database for matching fragmentation spectra (MS2) – followed by ChemSpider. For both databases the mass tolerance that the software used to search for matching mass peaks was set at 3 ppm. In the case of the mzCloud search the parameter "FT Fragment Mass Tolerance"was set to 5 ppm. The Human Metabolome Database (HMDB) was selected as the primary source for the ChemSpider search.
Ion Mode:	POSITIVE

MS ID:	MS003687
Analysis ID:	AN003952
Instrument Name:	Thermo Q Exactive Focus
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Full-MS scan spectra were acquired in the m/z range 75 – 1125, at a resolution of 70,000 (full width at half maximum (FWHM) at m/z 200) and 1×106 automatic gain control (AGC). MS/MS scan spectra were acquired at 17,500 resolution (FWHM at m/z 200), with 1×105 AGC, maximum injection time of 100 ms and dynamic exclusion of 6 s. Raw data files were independently processed by Compound Discoverer™ 3.1 (ThermoFisher Scientific) software for metabolomics data analysis. The preferred database used for metabolite identification was mzCloud – since the "Search mzCloud '' node searches this database for matching fragmentation spectra (MS2) – followed by ChemSpider. For both databases the mass tolerance that the software used to search for matching mass peaks was set at 3 ppm. In the case of the mzCloud search the parameter "FT Fragment Mass Tolerance"was set to 5 ppm. The Human Metabolome Database (HMDB) was selected as the primary source for the ChemSpider search.
Ion Mode:	NEGATIVE