Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA316309

Local Sample ID:	1_UT2
Subject ID:	SU003024
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	Seven- to eight-week-old female BALB/c mice
Gender:	Female
Animal Animal Supplier:	Jackson Laboratories

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Subject:

Subject ID:	SU003024
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	Seven- to eight-week-old female BALB/c mice
Gender:	Female
Animal Animal Supplier:	Jackson Laboratories

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
1_UT2	SA316309	FL037608	4-weeks infection	Factor

Collection:

Collection ID:	CO003017
Collection Summary:	Animals were divided into three different groups: healthy controls (Mtb-, n=6), infected mice four weeks post-infection (Mtb+4w, n=6) and infected mice twelve weeks post-infection (Mtb+12w, n=5). Seven- to eight-week-old female BALB/c mice were purchased from Jackson Laboratories. Mice were housed in a pathogen-free facility with ad libitum access to water and food. All the animal studies were performed following the guidelines approved by IACUC at the University of Alabama at Birmingham, USA. Mice were aerosol infected with Mtb H37Rv using the aerosol inhalation exposure system (Glas-Col, USA) to deliver ~120-250 CFU/mouse lung. The infection dose was estimated by enumerating the lung CFU at 24 hours post-infection. Mice were sacrificed using anaesthesia with isoflurane followed by gentle cervical dislocation. Mice organs were aseptically harvested and homogenized in 2 ml of 1x PBS, pH 7.4. Serial dilutions of homogenates were prepared in 1x PBS and plated on 7H11 agar plates supplemented with 10% ADS (Albumin, Dextrose, and NaCl), Carbenicillin (25 mg/L) and Cycloheximide (25 mg/L). Plates were incubated at 37 °C for ~21 days before counting colonies. Finally, lung samples were transferred to an N2(l)-containing recipient to freeze the tissues and stored at -80 °C to avoid postmortem metabolic processes.
Sample Type:	Lung

Treatment:

Treatment ID:	TR003033
Treatment Summary:	Animals were divided into three different groups: healthy controls (Mtb-, n=6), infected mice four weeks post-infection (Mtb+4w, n=6) and infected mice twelve weeks post-infection (Mtb+12w, n=5). Seven- to eight-week-old female BALB/c mice were purchased from Jackson Laboratories. Mice were housed in a pathogen-free facility with ad libitum access to water and food. All the animal studies were performed following the guidelines approved by IACUC at the University of Alabama at Birmingham, USA. Mice were aerosol infected with Mtb H37Rv using the aerosol inhalation exposure system (Glas-Col, USA) to deliver ~120-250 CFU/mouse lung. The infection dose was estimated by enumerating the lung CFU at 24 hours post-infection. Mice were sacrificed using anaesthesia with isoflurane followed by gentle cervical dislocation. Mice organs were aseptically harvested and homogenized in 2 ml of 1x PBS, pH 7.4. Serial dilutions of homogenates were prepared in 1x PBS and plated on 7H11 agar plates supplemented with 10% ADS (Albumin, Dextrose, and NaCl), Carbenicillin (25 mg/L) and Cycloheximide (25 mg/L). Plates were incubated at 37 °C for ~21 days before counting colonies. Finally, lung samples were transferred to an N2(l)-containing recipient to freeze the tissues and stored at -80 °C to avoid postmortem metabolic processes.

Treatment ID:

TR003033

Treatment Summary:

Animals were divided into three different groups: healthy controls (Mtb-, n=6), infected mice four weeks post-infection (Mtb+4w, n=6) and infected mice twelve weeks post-infection (Mtb+12w, n=5). Seven- to eight-week-old female BALB/c mice were purchased from Jackson Laboratories. Mice were housed in a pathogen-free facility with ad libitum access to water and food. All the animal studies were performed following the guidelines approved by IACUC at the University of Alabama at Birmingham, USA. Mice were aerosol infected with Mtb H37Rv using the aerosol inhalation exposure system (Glas-Col, USA) to deliver ~120-250 CFU/mouse lung. The infection dose was estimated by enumerating the lung CFU at 24 hours post-infection. Mice were sacrificed using anaesthesia with isoflurane followed by gentle cervical dislocation. Mice organs were aseptically harvested and homogenized in 2 ml of 1x PBS, pH 7.4. Serial dilutions of homogenates were prepared in 1x PBS and plated on 7H11 agar plates supplemented with 10% ADS (Albumin, Dextrose, and NaCl), Carbenicillin (25 mg/L) and Cycloheximide (25 mg/L). Plates were incubated at 37 °C for ~21 days before counting colonies. Finally, lung samples were transferred to an N2(l)-containing recipient to freeze the tissues and stored at -80 °C to avoid postmortem metabolic processes.

Sample Preparation:

Sampleprep ID:	SP003030
Sampleprep Summary:	The sample preparation and lipid extraction were performed at the Centers for AIDS Research and Free Radical Biology, University of Alabama at Birmingham (Birmingham, AL, United States), following a protocol initially described and optimized at CEMBIO (Madrid, Spain). Approximately 75 mg of lung tissue was mixed with a cold (–20°C) mixture of MeOH:H2O (1:1, v/v) added in a ratio of 1 mg tissue:10 µL of extraction solvent. Next, the tissue samples were homogenized using the Dounce homogenizer. After the homogenization, 200 µL of homogenate was mixed with 640 µL of MeOH and 160 µL of Methyl-Tert-Butyl ether (MTBE) to extract hydrophobic compounds. Samples were then vortex-mixed for 1 hour at room temperature (RT) and centrifuged at 4000 g for 20 min at 20°C. The samples were then passed through spin X columns (0.22 µm filter), and 200 µL of the filtered sample was dried at RT in the vacuum concentrator. From here, the samples were sent to CEMBIO for the UHPLC-MS analysis. Prior to the analysis, dried samples were re-suspended with 200 µL of MeOH/MTBE/H2O (7.4:1.6:1, v/v/v), which contained the corresponding ISs (C17 – sphingosine at 1 ppm for positive ion mode, and d31–palmitic acid at 3 ppm for negative ion mode). Samples were then centrifuged (16,100xg, 5 min, 15°C) before transferring them into sample vials with glass inserts for LC-MS analysis.

Sampleprep ID:

SP003030

Sampleprep Summary:

The sample preparation and lipid extraction were performed at the Centers for AIDS Research and Free Radical Biology, University of Alabama at Birmingham (Birmingham, AL, United States), following a protocol initially described and optimized at CEMBIO (Madrid, Spain). Approximately 75 mg of lung tissue was mixed with a cold (–20°C) mixture of MeOH:H2O (1:1, v/v) added in a ratio of 1 mg tissue:10 µL of extraction solvent. Next, the tissue samples were homogenized using the Dounce homogenizer. After the homogenization, 200 µL of homogenate was mixed with 640 µL of MeOH and 160 µL of Methyl-Tert-Butyl ether (MTBE) to extract hydrophobic compounds. Samples were then vortex-mixed for 1 hour at room temperature (RT) and centrifuged at 4000 g for 20 min at 20°C. The samples were then passed through spin X columns (0.22 µm filter), and 200 µL of the filtered sample was dried at RT in the vacuum concentrator. From here, the samples were sent to CEMBIO for the UHPLC-MS analysis. Prior to the analysis, dried samples were re-suspended with 200 µL of MeOH/MTBE/H2O (7.4:1.6:1, v/v/v), which contained the corresponding ISs (C17 – sphingosine at 1 ppm for positive ion mode, and d31–palmitic acid at 3 ppm for negative ion mode). Samples were then centrifuged (16,100xg, 5 min, 15°C) before transferring them into sample vials with glass inserts for LC-MS analysis.

Combined analysis:

Analysis ID	AN004780	AN004781
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Agilent 1290 Infinity II	Agilent 1290 Infinity II
Column	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Agilent 6545 QTOF	Agilent 6545 QTOF
Ion Mode	POSITIVE	NEGATIVE
Units	AREA	AREA

Chromatography:

Chromatography ID:	CH003612
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)
Column Temperature:	50 °C
Flow Gradient:	Started at 70% of B at 0 –1 min, 86% at 3.5 –10 min, and 100% B at 11–17 min
Flow Rate:	0.6 mL/min
Solvent A:	90% water/10% methanol; 10 mM ammonium acetate; 0.2 mM ammonium fluoride
Solvent B:	20% acetonitrile/30% methanol/50% isopropanol; 10 mM ammonium acetate; 0.2 mM ammonium fluoride
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004526
Analysis ID:	AN004780
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in positive and negative ESI modes in separate runs, operated in full scan mode from 40 to 1700 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds was infused throughout the whole analysis: purine (C5H4N4) at m/z 121.0509 and HP-0921 (C18H18O6N3P3F24) at m/z 922.0098. These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction.
Ion Mode:	POSITIVE

MS ID:	MS004527
Analysis ID:	AN004781
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in positive and negative ESI modes in separate runs, operated in full scan mode from 40 to 1700 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds was infused throughout the whole analysis: purine (C5H4N4) at m/z 119.0363 and HP-0921 (C18H18O6N3P3F24) at m/z 980.0163 (HP-0921 + acetate). These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction.
Ion Mode:	NEGATIVE