Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA123852

Local Sample ID:	15
Subject ID:	SU001524
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU001524
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
15	SA123852	FL014996	Agilent 6520 profile data	Data type

Collection:

Collection ID:	CO001519
Collection Summary:	Human serum was frozen at -80 C.
Sample Type:	Blood (serum)

Treatment:

Treatment ID:	TR001539
Treatment Summary:	See sampleprep

Sample Preparation:

Sampleprep ID:	SP001532
Sampleprep Summary:	4 mL frozen commercial pooled human serum (Innovative Research, Novi, MI, USA) was thawed at 4 °C, vortexed and aliquoted into 50 µL portions in 2 mL Eppendorf vials. Every 50 µL portion was mixed with 250 µL cold methanol and vortexed to precipitate proteins.38 After 20 min incubation at −20 °C, these mixtures were centrifuged at 20,800 x g for 10 min at 4 °C. The supernatants were transferred into clean 2.0 mL Eppendorf vials and then dried in an Eppendorf Vacufuge (Brinkmann Instruments, Westbury, NY, USA). The residue in each Eppendorf vial was reconstituted in 50 µL H2O:ACN (2:3 v/v), vortexed and centrifuged at 20,800 x g for 10 min at 4 °C. The supernatant in all Eppendorf vials was pooled into a 5 mL Eppendorf vial, vortexed and centrifuged at xx,xxx x g (To Dan: I should have used 5000 rpm or a typical rpm in the big centrifuge in the biosample prep lab) for 10 min at 4 °C to further remove any solid residue. The resultant supernatant was aliquoted into 50 µL portions in 1.5 mL Eppendorf vials and stored at −80 °C. Prior to repeated injections to LC-MS in this work, 8 portions were diluted to 200 µL each with H2O:ACN (2:3 v/v), pooled into a 2 mL LC vial and vortexed.

Sampleprep ID:

SP001532

Sampleprep Summary:

4 mL frozen commercial pooled human serum (Innovative Research, Novi, MI, USA) was thawed at 4 °C, vortexed and aliquoted into 50 µL portions in 2 mL Eppendorf vials. Every 50 µL portion was mixed with 250 µL cold methanol and vortexed to precipitate proteins.38 After 20 min incubation at −20 °C, these mixtures were centrifuged at 20,800 x g for 10 min at 4 °C. The supernatants were transferred into clean 2.0 mL Eppendorf vials and then dried in an Eppendorf Vacufuge (Brinkmann Instruments, Westbury, NY, USA). The residue in each Eppendorf vial was reconstituted in 50 µL H2O:ACN (2:3 v/v), vortexed and centrifuged at 20,800 x g for 10 min at 4 °C. The supernatant in all Eppendorf vials was pooled into a 5 mL Eppendorf vial, vortexed and centrifuged at xx,xxx x g (To Dan: I should have used 5000 rpm or a typical rpm in the big centrifuge in the biosample prep lab) for 10 min at 4 °C to further remove any solid residue. The resultant supernatant was aliquoted into 50 µL portions in 1.5 mL Eppendorf vials and stored at −80 °C. Prior to repeated injections to LC-MS in this work, 8 portions were diluted to 200 µL each with H2O:ACN (2:3 v/v), pooled into a 2 mL LC vial and vortexed.

Combined analysis:

Analysis ID	AN002421	AN002422	AN002423	AN002424
Analysis type	MS	MS	MS	MS
Chromatography type	HILIC	HILIC	HILIC	HILIC
Chromatography system	Agilent 1260 Infinity	Agilent 1260 Infinity	Agilent 1290 Infinity	Agilent 1290 Infinity
Column	Waters XBridge BEH Amide (15 cm x 2.1mm,2.5um)	Waters XBridge BEH Amide (15 cm x 2.1mm,2.5um)	Waters XBridge BEH Amide (15 cm x 2.1mm,2.5um)	Waters XBridge BEH Amide (15 cm x 2.1mm,2.5um)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	QTOF	QTOF	QTOF	QTOF
MS instrument name	Agilent 6520 QTOF	Agilent 6520 QTOF	Agilent 6545 QTOF	Agilent 6545 QTOF
Ion Mode	POSITIVE	POSITIVE	POSITIVE	POSITIVE
Units	Peak area	Peak area	Peak area	Peak area

Chromatography:

Chromatography ID:	CH001778
Chromatography Summary:	The mobile phase consisted of (A) H2O:ACN (95:5, v/v) with 5 mM ammonium acetate and 0.1% acetic acid, and (B) H2O:ACN (5:95, v/v), 5 mM ammonium acetate and 0.1% acetic acid. Gradient elution was performed as follows: 100% mobile phase B for 1.5 min, 100 to 78% B from 1.5-6.0 min, 78 to 50% B from 6.0-9.0 min, 50% B from 9.0-15.0 min, restoration to 100% B from 15.0-17.0 min, and continued 100% B from 17.0-30.0 to equilibrate the LC column. The flow rate was 0.3 mL/min, the injection volume was 5 µL, followed by an H2O:ACN (5:95, v/v) needle wash for 10 s, and the column temperature was 35 oC.
Instrument Name:	Agilent 1260 Infinity
Column Name:	Waters XBridge BEH Amide (15 cm x 2.1mm,2.5um)
Column Temperature:	35
Flow Gradient:	100% mobile phase B for 1.5 min, 100 to 78% B from 1.5-6.0 min, 78 to 50% B from 6.0-9.0 min, 50% B from 9.0-15.0 min, restoration to 100% B from 15.0-17.0 min, and continued 100% B from 17.0-30.0 to equilibrate the LC column.
Flow Rate:	0.3 mL/min
Solvent A:	95% water/5% acetonitrile; 0.1% acetic acid; 5 mM ammonium acetate
Solvent B:	5% water/95% acetonitrile; 0.1% acetic acid; 5 mM ammonium acetate
Chromatography Type:	HILIC

Chromatography ID:	CH001779
Chromatography Summary:	The mobile phase consisted of (A) H2O:ACN (95:5, v/v) with 5 mM ammonium acetate and 0.1% acetic acid, and (B) H2O:ACN (5:95, v/v), 5 mM ammonium acetate and 0.1% acetic acid. Gradient elution was performed as follows: 100% mobile phase B for 1.5 min, 100 to 78% B from 1.5-6.0 min, 78 to 50% B from 6.0-9.0 min, 50% B from 9.0-15.0 min, restoration to 100% B from 15.0-17.0 min, and continued 100% B from 17.0-30.0 to equilibrate the LC column. The flow rate was 0.3 mL/min, the injection volume was 5 µL, followed by an H2O:ACN (5:95, v/v) needle wash for 10 s, and the column temperature was 35 oC.
Instrument Name:	Agilent 1260 Infinity
Column Name:	Waters XBridge BEH Amide (15 cm x 2.1mm,2.5um)
Column Temperature:	35
Flow Gradient:	100% mobile phase B for 1.5 min, 100 to 78% B from 1.5-6.0 min, 78 to 50% B from 6.0-9.0 min, 50% B from 9.0-15.0 min, restoration to 100% B from 15.0-17.0 min, and continued 100% B from 17.0-30.0 to equilibrate the LC column.
Flow Rate:	0.3 mL/min
Solvent A:	95% water/5% acetonitrile; 0.1% acetic acid; 5 mM ammonium acetate
Solvent B:	5% water/95% acetonitrile; 0.1% acetic acid; 5 mM ammonium acetate
Chromatography Type:	HILIC

Chromatography ID:	CH001780
Chromatography Summary:	The mobile phase consisted of (A) H2O:ACN (95:5, v/v) with 5 mM ammonium acetate and 0.1% acetic acid, and (B) H2O:ACN (5:95, v/v), 5 mM ammonium acetate and 0.1% acetic acid. Gradient elution was performed as follows: 100% mobile phase B for 1.5 min, 100 to 78% B from 1.5-6.0 min, 78 to 50% B from 6.0-9.0 min, 50% B from 9.0-15.0 min, restoration to 100% B from 15.0-17.0 min, and continued 100% B from 17.0-30.0 to equilibrate the LC column. The flow rate was 0.3 mL/min, the injection volume was 5 µL, followed by an H2O:ACN (5:95, v/v) needle wash for 10 s, and the column temperature was 35 oC.
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters XBridge BEH Amide (15 cm x 2.1mm,2.5um)
Column Temperature:	35
Flow Gradient:	100% mobile phase B for 1.5 min, 100 to 78% B from 1.5-6.0 min, 78 to 50% B from 6.0-9.0 min, 50% B from 9.0-15.0 min, restoration to 100% B from 15.0-17.0 min, and continued 100% B from 17.0-30.0 to equilibrate the LC column.
Flow Rate:	0.3 mL/min
Solvent A:	95% water/5% acetonitrile; 0.1% acetic acid; 5 mM ammonium acetate
Solvent B:	5% water/95% acetonitrile; 0.1% acetic acid; 5 mM ammonium acetate
Chromatography Type:	HILIC

Chromatography ID:	CH001781
Chromatography Summary:	The mobile phase consisted of (A) H2O:ACN (95:5, v/v) with 5 mM ammonium acetate and 0.1% acetic acid, and (B) H2O:ACN (5:95, v/v), 5 mM ammonium acetate and 0.1% acetic acid. Gradient elution was performed as follows: 100% mobile phase B for 1.5 min, 100 to 78% B from 1.5-6.0 min, 78 to 50% B from 6.0-9.0 min, 50% B from 9.0-15.0 min, restoration to 100% B from 15.0-17.0 min, and continued 100% B from 17.0-30.0 to equilibrate the LC column. The flow rate was 0.3 mL/min, the injection volume was 5 µL, followed by an H2O:ACN (5:95, v/v) needle wash for 10 s, and the column temperature was 35 oC.
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters XBridge BEH Amide (15 cm x 2.1mm,2.5um)
Column Temperature:	35
Flow Gradient:	100% mobile phase B for 1.5 min, 100 to 78% B from 1.5-6.0 min, 78 to 50% B from 6.0-9.0 min, 50% B from 9.0-15.0 min, restoration to 100% B from 15.0-17.0 min, and continued 100% B from 17.0-30.0 to equilibrate the LC column.
Flow Rate:	0.3 mL/min
Solvent A:	95% water/5% acetonitrile; 0.1% acetic acid; 5 mM ammonium acetate
Solvent B:	5% water/95% acetonitrile; 0.1% acetic acid; 5 mM ammonium acetate
Chromatography Type:	HILIC

MS:

MS ID:	MS002258
Analysis ID:	AN002421
Instrument Name:	Agilent 6520 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The ESI conditions were as follows: Electrospray ion source ESI Agilent Jet Stream Technology in positive ionization mode; voltage 3.8 kV; desolvation temperature 325 °C; cone flow 20 L/h; desolvation gas flow 600 L/h; nebulizer pressure 45 psi, N2 drying gas; MS scan rate of 1.03 spectra/s across the range m/z 60-1000. The software, Progenesis Qi, was used to process raw data. In the results, every compound (feature) had at least two ions, like an ion and its isotope ion or adduct ion. Compounds with single ions were filtered out. If a compound had m/z information only, the m/z was directly used in the compound name. If a compound had neutral mass information, the neutral mass was converted to m/z.
Ion Mode:	POSITIVE

MS ID:	MS002259
Analysis ID:	AN002422
Instrument Name:	Agilent 6520 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The ESI conditions were as follows: Electrospray ion source ESI Agilent Jet Stream Technology in positive ionization mode; voltage 3.8 kV; desolvation temperature 325 °C; cone flow 20 L/h; desolvation gas flow 600 L/h; nebulizer pressure 45 psi, N2 drying gas; MS scan rate of 1.03 spectra/s across the range m/z 60-1000. The software, Progenesis Qi, was used to process raw data. In the results, every compound (feature) had at least two ions, like an ion and its isotope ion or adduct ion. Compounds with single ions were filtered out. If a compound had m/z information only, the m/z was directly used in the compound name. If a compound had neutral mass information, the neutral mass was converted to m/z.
Ion Mode:	POSITIVE

MS ID:	MS002260
Analysis ID:	AN002423
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	(Agilent 6545) MS scan rate of 1.03 spectra/s across the range m/z 60-1000. The software, Progenesis Qi, was used to process raw data. In the results, every compound (feature) had at least two ions, like an ion and its isotope ion or adduct ion. Compounds with single ions were filtered out. If a compound had m/z information only, the m/z was directly used in the compound name. If a compound had neutral mass information, the neutral mass was converted to m/z.
Ion Mode:	POSITIVE

MS ID:	MS002261
Analysis ID:	AN002424
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	(Agilent 6545) MS scan rate of 1.03 spectra/s across the range m/z 60-1000. The software, Progenesis Qi, was used to process raw data. In the results, every compound (feature) had at least two ions, like an ion and its isotope ion or adduct ion. Compounds with single ions were filtered out. If a compound had m/z information only, the m/z was directly used in the compound name. If a compound had neutral mass information, the neutral mass was converted to m/z.
Ion Mode:	POSITIVE