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MB Sample ID: SA177932

Local Sample ID:	UT3
Subject ID:	SU002000
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU002000
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
UT3	SA177932	FL022039	Untreated	Treatment
UT3	SA177932	FL022039	3	Donor

Collection:

Collection ID:	CO001993
Collection Summary:	PBMCs were isolated from healthy human blood cones (supplied by GlaxoSmithKlein) by gradient centrifugation. The PBMC layer was collected and monocytes isolated using CD14+ beads (Miltenyi Biotech) according to supplier’s protocol. For proteomics and metabolomics analysis, frozen human primary monocytes were used (supplied by Lonza). Purified monocytes were plated at relevant cell concentration for experiment and treated with growth factor GM-CSF (5 ng/ml) and cultured in RMPI-1640 (Life Technologies) with 5% FCS and 2mM L-glutamine for 6 days, at 37 °C, 5% CO2 to allow differentiation. All human biological samples were sourced ethically, and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol. On day 6, cells were washed once with treatment media RPMI-1640 with 2mM L-Glutamine and sensitised to complement attack by adding 7 µg/ml of anti-CD55, anti-CD59 and anti-HLA antibodies for 50 min at 37 °C, 5% CO2. Antibody-sensitised cells were exposed to normal human serum (NHS), or non-sensitised cells with NHS alone at 37 °C, 5% CO2 for the indicated amount of time. Subtlytic doses of MAC were characterised as <20% cell death (Campbell, Daw et al. 1979, Reid, Cooke et al. 2012). Alternatively, MAC attack was induced using human purified proteins C5b6-9 for the extracellular H2O2 assay. Cells were incubated with anti-CD59 for 50 min at 37 °C, 5% CO2. Antibody-sensitised cells were exposed to purified protein C5b6 for 10 min at room temperature, followed by addition of purified C7 for 15 min at 37 °C, 5% CO2. C8 and C9 were then added sequentially and left at 37 °C, 5% CO2 for the indicated amount of time. C7, C8 and C9 were added in a molar excess to the C5b6 concentration.
Sample Type:	human monocyte-derived macrophages (hMDM)

Treatment:

Treatment ID:	TR002012
Treatment Summary:	On day 6, cells were washed once with treatment media RPMI-1640 with 2mM L-Glutamine and sensitised to complement attack by adding 7 µg/ml of anti-CD55, anti-CD59 and anti-HLA antibodies for 50 min at 37 °C, 5% CO2. Antibody-sensitised cells were exposed to normal human serum (NHS), anti-C7 antibody with NHS (previously incubated for 30 min, on ice) as a negative control for MAC formation, or non-sensitised cells with NHS alone at 37 °C, 5% CO2 for the indicated amount of time. Subtlytic doses of MAC were characterised as <20% cell death (Campbell, Daw et al. 1979, Reid, Cooke et al. 2012). Alternatively, MAC attack was induced using human purified proteins C5b6-9 for the extracellular H2O2 assay. Cells were incubated with anti-CD59 for 50 min at 37 °C, 5% CO2. Antibody-sensitised cells were exposed to purified protein C5b6 for 10 min at room temperature, followed by addition of purified C7 for 15 min at 37 °C, 5% CO2. C8 and C9 were then added sequentially and left at 37 °C, 5% CO2 for the indicated amount of time. C7, C8 and C9 were added in a molar excess to the C5b6 concentration.

Treatment ID:

TR002012

Treatment Summary:

On day 6, cells were washed once with treatment media RPMI-1640 with 2mM L-Glutamine and sensitised to complement attack by adding 7 µg/ml of anti-CD55, anti-CD59 and anti-HLA antibodies for 50 min at 37 °C, 5% CO2. Antibody-sensitised cells were exposed to normal human serum (NHS), anti-C7 antibody with NHS (previously incubated for 30 min, on ice) as a negative control for MAC formation, or non-sensitised cells with NHS alone at 37 °C, 5% CO2 for the indicated amount of time. Subtlytic doses of MAC were characterised as <20% cell death (Campbell, Daw et al. 1979, Reid, Cooke et al. 2012). Alternatively, MAC attack was induced using human purified proteins C5b6-9 for the extracellular H2O2 assay. Cells were incubated with anti-CD59 for 50 min at 37 °C, 5% CO2. Antibody-sensitised cells were exposed to purified protein C5b6 for 10 min at room temperature, followed by addition of purified C7 for 15 min at 37 °C, 5% CO2. C8 and C9 were then added sequentially and left at 37 °C, 5% CO2 for the indicated amount of time. C7, C8 and C9 were added in a molar excess to the C5b6 concentration.

Sample Preparation:

Sampleprep ID:	SP002006
Sampleprep Summary:	hMDMs at 1 million cells/well in 24-well plates were sensitized to complement as usual and treated with a sublytic dose of NHS or left untreated for 4 hours. Following treatment, supernatants were removed and the hMDMs rinsed with fresh assay media (RPMI-1640 with 2mM L-Glutamine). For metabolite exaction, 75% 9:1 MeOH:CHCl3 was added directly to wells containing frozen cells (-80 °C). Cells from four technical replicates for each donor and sample type (NHS treated and untreated) were scraped from their respective wells, combined into Covaris adaptive focused acoustic tubes (for a total of 4 million cells per sample), and disrupted using a 2 minute lysis method on a Covaris S220 Focused Ulrasonicator (Peak Power - 200, Duty Factor - 10, Cycle/Burst - 200). Lysed samples were centrifuged at 5000 x g for 15 minutes at room temperature. Supernatant was split into two equal fractions and set on a Speedvac concentrator to dryness. Samples were reconstituted in either 3:1 MeCN:H2O or H2O+0.1% formic acid for analysis by LC-MS/MS with hydrophilic interaction (HILIC) or reverse phase chromatography respectively.

Sampleprep ID:

SP002006

Sampleprep Summary:

hMDMs at 1 million cells/well in 24-well plates were sensitized to complement as usual and treated with a sublytic dose of NHS or left untreated for 4 hours. Following treatment, supernatants were removed and the hMDMs rinsed with fresh assay media (RPMI-1640 with 2mM L-Glutamine). For metabolite exaction, 75% 9:1 MeOH:CHCl3 was added directly to wells containing frozen cells (-80 °C). Cells from four technical replicates for each donor and sample type (NHS treated and untreated) were scraped from their respective wells, combined into Covaris adaptive focused acoustic tubes (for a total of 4 million cells per sample), and disrupted using a 2 minute lysis method on a Covaris S220 Focused Ulrasonicator (Peak Power - 200, Duty Factor - 10, Cycle/Burst - 200). Lysed samples were centrifuged at 5000 x g for 15 minutes at room temperature. Supernatant was split into two equal fractions and set on a Speedvac concentrator to dryness. Samples were reconstituted in either 3:1 MeCN:H2O or H2O+0.1% formic acid for analysis by LC-MS/MS with hydrophilic interaction (HILIC) or reverse phase chromatography respectively.

Combined analysis:

Analysis ID	AN003123	AN003124
Analysis type	MS	MS
Chromatography type	HILIC	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000 RS	Thermo Dionex Ultimate 3000 RS
Column	Phenomenex Luna NH2 (150 x 2.1mm,3um)	Waters Acquity BEH C18 (150 x 2mm,1.7um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	UNSPECIFIED	POSITIVE
Units	counts	counts

Chromatography:

Chromatography ID:	CH002308
Chromatography Summary:	For HILIC LC-MS/MS analysis, samples were analysed in positive and negative ion mode using a Phenomenex Luna NH2 analytical column (100 mm x 2 mm, 3 µm) held at room temperature with 10 minute linear gradient (A - 5% MeCN/20mM NH4CH2CO2/20mM NH4OH; B - MeCN) from 95 to 0 % MeCN followed by 5 minute hold at a flow rate of 0.400 mL/min.
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Phenomenex Luna NH2 (150 x 2.1mm,3um)
Chromatography Type:	HILIC

Chromatography ID:	CH002309
Chromatography Summary:	For reverse phase LC-MS/MS analysis, samples were analysed in positive ion mode only using a Waters Acquity BEH C18 analytical column (100 mm x 2.1 mm, 1.7 µm) held at 40oC with 4 minute linear gradient (A - H2O + 0.1% formic acid; B - MeOH + 0.1% formic acid) from 0.5 to 70% MeOH followed by a 5 minute hold at a flow rate of 0.350 mL/min.
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Waters Acquity BEH C18 (150 x 2mm,1.7um)
Column Temperature:	40
Flow Gradient:	4 minute linear gradient from 0.5 to 70% MeOH followed by a 5 minute hold
Flow Rate:	0.350 mL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% methanol; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002904
Analysis ID:	AN003123
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Mass spectrometric analysis was performed using data dependent acquisition. Full scan spectra were acquired at a scan range of 61 to 915 m/z at a resolution of 70,000 with an automatic gain control (AGC) of 1e6 ions and maximum injection time of 200 ms. Top 7 data dependent acquisition was employed with priority placed on a custom inclusion list built for known metabolite features. The custom inclusion list was derived from the analysis of neat standards part of the Mass Spectrometry Metabolite Library (IROA Technologies). Precursor ions were isolated with a quadrupole mass window of 2.0 m/z and collision induced dissociation fragmentation performed with stepped collision energy of 20, 30, and 45 V. MS/MS spectra were acquired at a resolution of 17,500 with an AGC target of 3e3 ions and a maximum injection time of 200 ms. Raw data was aligned, integrated, and grouped using Thermo Compound Discoverer v3.1.0.305. Deuterated L-Tryptophan, L-Phenylalanine, and Caffeine internal standards added to sample reconstitution solvents were used for data normalization. Peak annotation was based on the same database used to build the custom inclusion list for known metabolite features, and included retention time, m/z, and MS/MS data for > 500 primary metabolites. Peaks not annotated using the custom database were searched against m/z Cloud as an alternative approach to peak annotation. Statistical and pathway enrichment analysis, as well as data representation was performed using MetaboAnalyst 5.0 (Xia and Wishart 2010, Pang, Chong et al. 2021).
Ion Mode:	UNSPECIFIED

MS ID:	MS002905
Analysis ID:	AN003124
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Mass spectrometric analysis was performed using data dependent acquisition. Full scan spectra were acquired at a scan range of 61 to 915 m/z at a resolution of 70,000 with an automatic gain control (AGC) of 1e6 ions and maximum injection time of 200 ms. Top 7 data dependent acquisition was employed with priority placed on a custom inclusion list built for known metabolite features. The custom inclusion list was derived from the analysis of neat standards part of the Mass Spectrometry Metabolite Library (IROA Technologies). Precursor ions were isolated with a quadrupole mass window of 2.0 m/z and collision induced dissociation fragmentation performed with stepped collision energy of 20, 30, and 45 V. MS/MS spectra were acquired at a resolution of 17,500 with an AGC target of 3e3 ions and a maximum injection time of 200 ms. Raw data was aligned, integrated, and grouped using Thermo Compound Discoverer v3.1.0.305. Deuterated L-Tryptophan, L-Phenylalanine, and Caffeine internal standards added to sample reconstitution solvents were used for data normalization. Peak annotation was based on the same database used to build the custom inclusion list for known metabolite features, and included retention time, m/z, and MS/MS data for > 500 primary metabolites. Peaks not annotated using the custom database were searched against m/z Cloud as an alternative approach to peak annotation. Statistical and pathway enrichment analysis, as well as data representation was performed using MetaboAnalyst 5.0 (Xia and Wishart 2010, Pang, Chong et al. 2021).
Ion Mode:	POSITIVE