Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA212963

Local Sample ID:	PL244_1233887
Subject ID:	SU002321
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	50 +/-11
Gender:	Female

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Subject:

Subject ID:	SU002321
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	50 +/-11
Gender:	Female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
PL244_1233887	SA212963	FL025980	Group 1	Factor

Collection:

Collection ID:	CO002314
Collection Summary:	EDTA plasma from cancer-free women (n = 167) were obtained from the MD Anderson Cancer Center (MDACC) Longitudinal High-Risk Cohort initiated September 1st, 2011, for the prospective follow-up of cancer-free highrisk women seen in the MDACC Cancer Prevention Center (IRB protocol LAB07-0086).
Sample Type:	Blood (plasma)

Treatment:

Treatment ID:	TR002333
Treatment Summary:	The specimen set consisted of pre-treatment EDTA plasma from 88 patients who received standard-of-care NACT; 62 of the 88 patients had a second plasma sample available after four cycles of AC.

Sample Preparation:

Sampleprep ID:	SP002327
Sampleprep Summary:	Plasma metabolites were extracted from pre-aliquoted biospecimens (15µL) with 45µL of LCMS grade methanol (ThermoFisher) in a 96-well microplate (Eppendorf). Plates were heat sealed, vortexed for 5 min at 750 rpm, and centrifuged at 2000 × g for 10 minutes at room temperature. The supernatant (30µL) was transferred to a 96-well plate, leaving behind the precipitated protein. The supernatant was further diluted with 60µL of 100mM ammonium formate, pH3 (Fisher Scientific). For Hydrophilic Interaction Liquid Chromatography (HILIC) positive ion analysis, 15µL of the supernatant and ammonium formate mix were diluted with 195µL of 1:3:8:144 water (GenPure ultrapure water system, Thermofisher): LCMS grade methanol (ThermoFisher): 100mM ammonium formate, pH3 (Fisher Scientific): LCMS grade acetonitrile (ThermoFisher). For C18 analysis, 15µL of the supernatant and ammonium formate mix were diluted with 90µL water (GenPure ultrapure water system, ThermoFisher) for positive ion mode. Each sample solution was transferred to 384-well microplate (Eppendorf) for LCMS analysis.

Sampleprep ID:

SP002327

Sampleprep Summary:

Plasma metabolites were extracted from pre-aliquoted biospecimens (15µL) with 45µL of LCMS grade methanol (ThermoFisher) in a 96-well microplate (Eppendorf). Plates were heat sealed, vortexed for 5 min at 750 rpm, and centrifuged at 2000 × g for 10 minutes at room temperature. The supernatant (30µL) was transferred to a 96-well plate, leaving behind the precipitated protein. The supernatant was further diluted with 60µL of 100mM ammonium formate, pH3 (Fisher Scientific). For Hydrophilic Interaction Liquid Chromatography (HILIC) positive ion analysis, 15µL of the supernatant and ammonium formate mix were diluted with 195µL of 1:3:8:144 water (GenPure ultrapure water system, Thermofisher): LCMS grade methanol (ThermoFisher): 100mM ammonium formate, pH3 (Fisher Scientific): LCMS grade acetonitrile (ThermoFisher). For C18 analysis, 15µL of the supernatant and ammonium formate mix were diluted with 90µL water (GenPure ultrapure water system, ThermoFisher) for positive ion mode. Each sample solution was transferred to 384-well microplate (Eppendorf) for LCMS analysis.

Combined analysis:

Analysis ID	AN003645
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters XEVO-G2XSQTOF
Column	C18
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Waters Xevo-G2-XS
Ion Mode	POSITIVE
Units	ppm

Chromatography:

Chromatography ID:	CH002699
Chromatography Summary:	Instrument: XEVO-G2XSQTOF Untargeted metabolomics analysis was conducted on Waters Acquity™ UPLC system with 2D column regeneration configuration (I-class and H-class) coupled to a Xevo G2-XS quadrupole time-of-flight (qTOF) mass spectrometer as previously described.11,13-15 Chromatographic separation was performed using HILIC (Acquity™ UPLC BEH amide, 100 Å, 1.7 µm 2.1× 100mm, Waters Corporation, Milford, U.S.A) and C18 (Acquity™ UPLC HSS T3, 100 Å, 1.8 µm, 2.1×100mm, Water Corporation, Milford, U.S.A) columns at 45°C. Quaternary solvent system mobile phases were (A) 0.1% formic acid in water, (B) 0.1% formic acid in acetonitrile and (D) 100mM ammonium formate, pH 3. Samples were separated on the HILIC using the following gradient profile at 0.4 mL/min flow rate: (95% B, 5% D) linear change to (70% A, 25% B and 5% D) over 5 min; 100% A for 1 min; and 100% A for 1 min. For C18 separation, the chromatography gradient was as follows at 0.4 mL/min flow rate: 100% A with a linear change to (5% A, 95% B) over 5 min; (95% B, 5% D) for 1 min; and 1 min at (95% B, 5% D). A binary pump was used for column regeneration and equilibration. The solvent system mobile phases were (A1) 100mM ammonium formate, pH 3, (A2) 0.1% formic in 2-propanol and (B1) 0.1% formic acid in acetonitrile. The HILIC column was stripped using 90% A2 for 5 min at 0.25 mL/min flow rate, followed by a 2 min equilibration using 100% B1 at 0.3mL/min flow rate. Reverse phase C18 column regeneration was performed using 95% A1, 5% B1 for 2 min followed by column equilibration using 5% A1, 95% B1 for 5 min at 0.4mL/min flow rate.
Instrument Name:	Waters XEVO-G2XSQTOF
Column Name:	C18
Column Temperature:	45
Flow Gradient:	100% A with a linear change to (5% A, 95% B) over 5 min; (95% B, 5% D) for 1 min; and 1 min at (95% B, 5% D)
Flow Rate:	0.4 mL/min
Solvent A:	100% water; 0.1% formic acid(A), 100% acetonitrile; 0.1% formic acid(B), 100 mM ammonium formate, pH 3(D)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003396
Analysis ID:	AN003645
Instrument Name:	Waters Xevo-G2-XS
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Data were processed using Progenesis QI (Non-linear, Waters). Peak picking and retention time alignment of LC-MS and MSe data were performed using Progenesis QI software (Non-linear, Waters)
Ion Mode:	POSITIVE