Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA081230

Local Sample ID:	82
Subject ID:	SU001237
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Female

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Subject:

Subject ID:	SU001237
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
82	SA081230	FL012390	UT	Group

Collection:

Collection ID:	CO001231
Collection Summary:	Blood samples were collected via retro-orbital bleeding after anesthesia, from 22 TKO-Pre mice (average age, 2.0 months; age range, 1.3–3.1), 10 TKO-ET mice (4.8 months: 2.5–5.9), 16 TKO-AT mice (6.4 months: 4.3–10.1), 19 TKO-ctrl mice (3.0 months: 3.0–8.2), and 17 UT mice (5.4 months: 3.7–6.8). Blood samples were centrifuged at 14,000 rpm for 5 minutes at room temperature, and serum was collected and stored at -80 °C until UPLC-MS analysis.
Sample Type:	Blood (serum)

Treatment:

Treatment ID:	TR001252
Treatment Summary:	Triple-mutant (TKO) mice (p53 LSL-R172H/+ Dicer1 flox/flox Pten flox/flox Amhr2 cre/+) were generated by mating p53 LSL-R172H/+ Dicer1 flox/flox Pten flox/flox female with Dicer1 flox/flox Pten flox/flox Amhr2 cre/+ male mice. Serum samples were collected from TKO mice at different stages of tumor progression: from premalignant stage to early stage to advanced stage. Precursor lesions are fallopian tubes at premalignant stage, when no tumors are yet present. TKO mice with precursor (i.e. premalignant stage) lesions in the fallopian tubes are referred to as “TKO-Pre”; TKO-ET (early tumor) are TKO mice with early-stage tumors in the fallopian tube without evidence of metastasis; TKO-AT (advanced-stage tumor) are TKO mice with ovarian and peritoneal metastases accompanied by ascites; and TKO-ctrl are control mice (p53 LSL-R172H/+ Dicer1 flox/flox Pten flox/flox), which have the same genetic background as TKO mice but develop no tumors. All TKO mice developed high-grade serous carcinoma (HGSC, i.e. high-grade serous ovarian cancer).

Treatment ID:

TR001252

Treatment Summary:

Triple-mutant (TKO) mice (p53 LSL-R172H/+ Dicer1 flox/flox Pten flox/flox Amhr2 cre/+) were generated by mating p53 LSL-R172H/+ Dicer1 flox/flox Pten flox/flox female with Dicer1 flox/flox Pten flox/flox Amhr2 cre/+ male mice. Serum samples were collected from TKO mice at different stages of tumor progression: from premalignant stage to early stage to advanced stage. Precursor lesions are fallopian tubes at premalignant stage, when no tumors are yet present. TKO mice with precursor (i.e. premalignant stage) lesions in the fallopian tubes are referred to as “TKO-Pre”; TKO-ET (early tumor) are TKO mice with early-stage tumors in the fallopian tube without evidence of metastasis; TKO-AT (advanced-stage tumor) are TKO mice with ovarian and peritoneal metastases accompanied by ascites; and TKO-ctrl are control mice (p53 LSL-R172H/+ Dicer1 flox/flox Pten flox/flox), which have the same genetic background as TKO mice but develop no tumors. All TKO mice developed high-grade serous carcinoma (HGSC, i.e. high-grade serous ovarian cancer).

Sample Preparation:

Sampleprep ID:	SP001245
Sampleprep Summary:	Serum samples were thawed on ice and subject to two different sample preparation protocols to profile either non-polar or polar sub-metabolomes. Reverse phase (RP) and hydrophilic interaction liquid chromatography (HILIC) UPLC-MS analysis were used for these different polarity metabolite fractions to obtain complementary information. Methanol (for polar) or iso-propanol (for non-polar) was added to a 50 μL serum sample in a 3:1 ratio to precipitate proteins. Samples were vortex mixed for 10 s and centrifuged at 13,000 rpm for 7 min. Then, 150 μL supernatant was frozen at -80 °C for UPLC-MS analysis. A sample blank was prepared with 50 μL of LC-MS grade water, and a pooled quality control (QC) sample was created by mixing 10 μL aliquot of each serum sample. Both the sample blank and the pooled sample were processed with the same procedure as the murine serum samples. Solvent blanks and sample blanks were analyzed together with murine serum samples.

Sampleprep ID:

SP001245

Sampleprep Summary:

Serum samples were thawed on ice and subject to two different sample preparation protocols to profile either non-polar or polar sub-metabolomes. Reverse phase (RP) and hydrophilic interaction liquid chromatography (HILIC) UPLC-MS analysis were used for these different polarity metabolite fractions to obtain complementary information. Methanol (for polar) or iso-propanol (for non-polar) was added to a 50 μL serum sample in a 3:1 ratio to precipitate proteins. Samples were vortex mixed for 10 s and centrifuged at 13,000 rpm for 7 min. Then, 150 μL supernatant was frozen at -80 °C for UPLC-MS analysis. A sample blank was prepared with 50 μL of LC-MS grade water, and a pooled quality control (QC) sample was created by mixing 10 μL aliquot of each serum sample. Both the sample blank and the pooled sample were processed with the same procedure as the murine serum samples. Solvent blanks and sample blanks were analyzed together with murine serum samples.

Combined analysis:

Analysis ID	AN001937	AN001938	AN001939	AN001940
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	HILIC	HILIC
Chromatography system	Thermo Dionex Ultimate 3000	Thermo Dionex Ultimate 3000	Thermo Dionex Ultimate 3000	Thermo Dionex Ultimate 3000
Column	Waters Acquity BEH C18 (50 x 2.1mm,1.7um)	Waters Acquity BEH C18 (50 x 2.1mm,1.7um)	Waters Acquity BEH HILIC (150 x 2.1mm,1.7um)	Waters Acquity BEH HILIC (150 x 2.1mm,1.7um)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	chromatographic peak area	chromatographic peak area	chromatographic peak area	chromatographic peak area

Chromatography:

Chromatography ID:	CH001406
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Waters Acquity BEH C18 (50 x 2.1mm,1.7um)
Column Temperature:	55
Sample Injection:	5μL
Solvent A:	60% acetonitrile/40% water; 0.1% formic acid; 10 mM ammonium formate
Solvent B:	90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate
Chromatography Type:	Reversed phase

Chromatography ID:	CH001407
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Waters Acquity BEH HILIC (150 x 2.1mm,1.7um)
Column Temperature:	55
Sample Injection:	2μL
Solvent A:	95% water/5% acetonitrile; 0.05% ammonium hydroxide; 10 mM ammonium acetate
Solvent B:	100% acetonitrile; 0.05% ammonium hydroxide
Chromatography Type:	HILIC

MS:

MS ID:	MS001793
Analysis ID:	AN001937
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Samples were analyzed in positive and negative ion modes for both RP and HILIC chromatography. Spectral features were extracted from the raw data using Compound Discoverer v2.1 software.
Ion Mode:	POSITIVE
Capillary Temperature:	275 °C
Spray Voltage:	3.0 kV

MS ID:	MS001794
Analysis ID:	AN001938
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Samples were analyzed in positive and negative ion modes for both RP and HILIC chromatography. Spectral features were extracted from the raw data using Compound Discoverer v2.1 software.
Ion Mode:	NEGATIVE
Capillary Temperature:	275 °C
Spray Voltage:	-2.8 kV

MS ID:	MS001795
Analysis ID:	AN001939
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Samples were analyzed in positive and negative ion modes for both RP and HILIC chromatography. Spectral features were extracted from the raw data using Compound Discoverer v2.1 software.
Ion Mode:	POSITIVE
Capillary Temperature:	275 °C
Spray Voltage:	3.0 kV

MS ID:	MS001796
Analysis ID:	AN001940
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Samples were analyzed in positive and negative ion modes for both RP and HILIC chromatography. Spectral features were extracted from the raw data using Compound Discoverer v2.1 software.
Ion Mode:	NEGATIVE
Capillary Temperature:	275 °C
Spray Voltage:	-2.8 kV