Return to study ST001328 main page

MB Sample ID: SA096367

Local Sample ID:GCMS_4W1
Subject ID:SU001402
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6
Age Or Age Range:8-10 weeks old
Gender:Male and female
Animal Feed:ad libitum
Animal Water:ad libitum

Select appropriate tab below to view additional metadata details:


Subject:

Subject ID:SU001402
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6
Age Or Age Range:8-10 weeks old
Gender:Male and female
Animal Feed:ad libitum
Animal Water:ad libitum

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
GCMS_4W1SA096367FL0135964 weeks post-infectionPulmonary TB status

Collection:

Collection ID:CO001397
Collection Summary:Both male and female age-matched C57BL/6 mice (8-10 weeks old) were infected with Mtb H37Rv in animal BSL-3 laboratory and monitored with food and water ad libitum. Mice were sacrificed by anesthesia with isoflurane followed by gentle cervical dislocation as approved by institutional Animal Protocol Number (APN): 08591. Mice experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Alabama at Birmingham, USA. For mice studies, we adhered as per national/international regulation of “Public Health Service Policy on Humane Care and Use of Laboratory Animals” (NIH), and “Animal Welfare Act and Animal Welfare Regulations” (USDA). Mouse genotype was confirmed by PCR and Western blotting. Mtb H37Rv was grown at 37 °C with shaking in BD Difco Middlebrook 7H9 media supplemented with 0.2 % glycerol, ADS (Albumin, Dextrose, NaCl) with 0.02 % tyloxapol. Mice were infected with 5 x 104 Mtb H37Rv via the intratracheal route. Lungs were collected from uninfected (n = 4), and Mtb-infected mice at 4- and 9-weeks post-infection (n = 4 and n = 5, respectively), and stored immediately at -80 °C for further processing and metabolite extraction.
Sample Type:Lung
Storage Conditions:-80℃

Treatment:

Treatment ID:TR001417
Treatment Summary:Mice were infected with 5 x 104 Mtb H37Rv via the intratracheal route and zero timepoint and monitored for disease progression until lung collection at 4 or 9 weeks post-infection
Treatment:Infection
Treatment Route:Intratracheal
Treatment Dose:5 x 10^4 Mtb H37Rv

Sample Preparation:

Sampleprep ID:SP001410
Sampleprep Summary:After lung collection, 1 mL of 50 % methanol was added to 100 mg of Mtb-infected or uninfected lung tissue and homogenized in a dounce homogenizer to prepare a uniform suspension. For CE-TOF/MS, 200 uL of homogenate were mixed with 200 L of 0.2 M formic acid and vortexed for 2 min. The samples were cleared by centrifugation at 16000 x g for 10 min at 4 °C and the supernatant was filter-sterilized using 0.22 um spin-X columns (Sigma). For GC-QTOF/MS and LC-QTOF/MS, 200 uL of each sample homogenate were mixed with 800 L of 80:20 methanol:MTBE (Methyl tert-butyl ether) and vortexed for 2 min. Metabolites were then extracted for 1 h with shaking at room temperature and then centrifuged at 4000 x g at 20 °C for 20 min. Supernatants were sterile filtered using 0.22 um Spin-X columns. All samples were passed through a Millipore filter (30-kDa cutoff) to remove large proteins. Samples were dried under high vacuum and stored at -80 °C until further platform-specific processing and analysis. For CE-ESI(+)-TOF/MS analysis, The dried samples were resuspended in Milli-Q water containing 0.1 mM formic acid and 0.2 mM methionine sulfone (internal standard) (Sigma-Aldrich, Germany) by vortexing for 1 min. After subsequent centrifugation (12600 x g, 15 min), the resulting clear solution was analyzed. For GC-QTOF/MS analysis, The above described dried samples were re-suspended in 450 µL of MeOH:H2O:MTBE (74:10:16) and after centrifugation at 12600 x g, 15 min at 4 °C, the supernatant was transferred to a vial with insert and evaporated to dryness under high vacuum. The obtained dried extracts were derivatized by a MPS autosampler for GC/MS analysis as previously described (Fiehn O., 2006). For LC-QTOF/MS analysis, The above described dried samples were resuspended in 200 µL of methanol:water:MTBE (7.4:1:1.6), vortexed for 1.5 h and centrifuged (4000 x g, 10 min, 4 °C). Clear solutions were analyzed.
Sample Derivatization:In GC-EI-QTOF/MS analyses, Briefly, aldehyde and keto groups were first converted to O-methyloximes by reaction with 10 µL pyridine containing 15 mg/mL O-methoxyamine (Sigma-Aldrich, Germany) for 60 min at 70 °C. In a second step, acid hydrogen-containing metabolites were trimethylsilylated by reaction with 10 µL N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) (Sigma-Aldrich, Germany) to enhance the GC/MS metabolite coverage

Combined analysis:

Analysis ID AN002211 AN002212 AN002213 AN002214
Analysis type MS MS MS MS
Chromatography type Reversed phase Reversed phase GC CE
Chromatography system Agilent 1200 Agilent 1200 Agilent 7890B Agilent 7100 CE
Column Agilent Poroshell 120 EC-C8 (150 x 2.1mm,2.7um) Agilent Poroshell 120 EC-C8 (150 x 2.1mm,2.7um) Agilent DB5-MS (30m x 0.25mm, 0.25um) Agilent bare-fused silica capillary (96 cm x 50 um i.d.)
MS Type ESI ESI EI ESI
MS instrument type QTOF QTOF QTOF TOF
MS instrument name Agilent 6520 QTOF Agilent 6520 QTOF Agilent 7200 QTOF Agilent 6224 TOF
Ion Mode POSITIVE NEGATIVE POSITIVE POSITIVE
Units Abundance Abundance Abundance Abundance

Chromatography:

Chromatography ID:CH001621
Chromatography Summary:5 μL of extracted lung samples were injected into a thermostated (60 °C) Agilent Poroshell 120 EC-C8 column (150 mm × 2.1 mm, 2.7 μm; Agilent Technologies, CA, USA) with a guard column Ascentis Express C8 (5 mm × 2.1 mm, 2.7 μm; Supelco, Bellefonte, PA, USA). The flow rate was 0.4 mL·min-1 with solvent A (10 mM ammonium formate in Milli-Q water) and solvent B (10 mM ammonium formate in methanol and 15 % of isopropanol) for analysis in positive ionization mode. Initial conditions at time 0 were 82 % B, increasing to 96 % B in 30 min. This was then held until 38 min. The gradient then increased to 100 % B by 38.5 min and held until 40.5 min. The conditions were then returned to the starting conditions by 42 min, followed by an 8 min re-equilibration time. The total run time of the method was 50 min.
Instrument Name:Agilent 1200
Column Name:Agilent Poroshell 120 EC-C8 (150 x 2.1mm,2.7um)
Column Temperature:60
Flow Gradient:Initial conditions at time 0 were 82 % B, increasing to 96 % B in 30 min. This was then held until 38 min. The gradient then increased to 100 % B by 38.5 min and held until 40.5 min. The conditions were then returned to the starting conditions by 42 min, followed by an 8 min re-equilibration time. The total run time of the method was 50 min.
Flow Rate:0.4mL·min-1
Solvent A:100% water; 0.1% formic acid
Solvent B:85% methanol/15% isopropanol; 0.1 % formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH001622
Chromatography Summary:5 μL of extracted lung samples were injected into a thermostated (60 °C) Agilent Poroshell 120 EC-C8 column (150 mm × 2.1 mm, 2.7 μm; Agilent Technologies, CA, USA) with a guard column Ascentis Express C8 (5 mm × 2.1 mm, 2.7 μm; Supelco, Bellefonte, PA, USA). The flow rate was 0.4 mL·min-1 with solvent A (MilliQ water with 0.1 % formic acid), and solvent B (methanol with 0.1 % formic acid and 15 % of isopropanol) for analysis in negative ionization mode. Initial conditions at time 0 were 82 % B, increasing to 96 % B in 30 min. This was then held until 38 min. The gradient then increased to 100 % B by 38.5 min and held until 40.5 min. The conditions were then returned to the starting conditions by 42 min, followed by an 8 min re-equilibration time. The total run time of the method was 50 min.
Instrument Name:Agilent 1200
Column Name:Agilent Poroshell 120 EC-C8 (150 x 2.1mm,2.7um)
Column Temperature:60
Flow Gradient:Initial conditions at time 0 were 82 % B, increasing to 96 % B in 30 min. This was then held until 38 min. The gradient then increased to 100 % B by 38.5 min and held until 40.5 min. The conditions were then returned to the starting conditions by 42 min, followed by an 8 min re-equilibration time. The total run time of the method was 50 min.
Flow Rate:0.4mL·min-1
Solvent A:100% water; 0.1% formic acid
Solvent B:85% methanol/15% isopropanol; 0.1 % formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH001623
Chromatography Summary:1 µL of sample was injected into a multimode inlet at 230 °C with a split ratio set at 1:12 with 9.354 mL·min-1 connected to a capillary column (30 m x 0.25 mm x 0.25 µm; Agilent, Germany). Helium was used as carrier gas, at a flow rate of 0.78 mL·min-1. Column temperature was 60 °C for 1 min and then programmed to increase at a rate of 10 °C·min-1 until 325 °C which was maintained for 10 min. Total runtime was 37.5 min.
Instrument Name:Agilent 7890B
Column Name:Agilent DB5-MS (30m x 0.25mm, 0.25um)
Flow Rate:9.354 mL·min-1
Chromatography Type:GC
  
Chromatography ID:CH001624
Chromatography Summary:The separation occurred in a fused-silica capillary (Agilent Technologies) (total length, 96 cm; i.d., 50 μm) under normal polarity with a background electrolyte containing 1.0 M formic acid in 10 % (v/v) methanol at 20 °C. Sheath liquid (6 µL·min-1) was methanol/water (1/1, v/v) containing 1.0 mM formic acid with two reference masses to allow correction and high mass resolution in the MS. Samples were hydrodynamically injected at 50 mbar for 35 s and stacked by injecting background electrolyte at 100 mbar for 10 s.
Instrument Name:Agilent 7100 CE
Column Name:Agilent bare-fused silica capillary (96 cm x 50 um i.d.)
Chromatography Type:CE

MS:

MS ID:MS002057
Analysis ID:AN002211
Instrument Name:Agilent 6520 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:Mass spectrometry detection was performed in positive ESI mode in a full scan from 100 to 1000 m/z. Samples were analyzed in separate runs (positive and negative ionization modes), in a randomized order. Capillary voltage was set to 4.5 kV; the drying gas flow rate was 10 L·min-1 at 350 °C and gas nebulizer at 40 psi; fragmentor voltage, skimmer voltage and octopole radio frequency voltage were set to 175, 65 and 750 V, respectively. Data were collected at a scan rate of 1.05 spectra·s-1. The resulting LC-QTOF/MS data files were cleaned of background noise and unrelated ions by the Batch Recursive Feature Extraction tool with Agilent MassHunter Profinder software version B.06.00. Data were extracted using data mining algorithms of the software.
Ion Mode:POSITIVE
Capillary Voltage:4.5 kV
Dry Gas Flow:10 L·min-1
Dry Gas Temp:350 °C
Gas Pressure:40 psi
Octpole Voltage:750 V
  
MS ID:MS002058
Analysis ID:AN002212
Instrument Name:Agilent 6520 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:Mass spectrometry detection was performed in negative ESI mode in a full scan from 100 to 1000 m/z. Samples were analyzed in separate runs (positive and negative ionization modes), in a randomized order. Capillary voltage was set to 4.5 kV; the drying gas flow rate was 10 L·min-1 at 350 °C and gas nebulizer at 40 psi; fragmentor voltage, skimmer voltage and octopole radio frequency voltage were set to 175, 65 and 750 V, respectively. Data were collected at a scan rate of 1.05 spectra·s-1. The resulting LC-QTOF/MS data files were cleaned of background noise and unrelated ions by the Batch Recursive Feature Extraction tool with Agilent MassHunter Profinder software version B.06.00. Data were extracted using data mining algorithms of the software.
Ion Mode:NEGATIVE
Capillary Voltage:4.5 kV
Dry Gas Flow:10 L·min-1
Dry Gas Temp:350 °C
Gas Pressure:40 psi
Octpole Voltage:750 V
  
MS ID:MS002059
Analysis ID:AN002213
Instrument Name:Agilent 7200 QTOF
Instrument Type:QTOF
MS Type:EI
MS Comments:The analysis was performed on an Agilent Technologies 7200 accurate mass Q/TOF analyzer equipped with an electron ionization (EI) source.The MS scan mode was chosen as the acquisition mode, with the mass range of 50-650 m/z and an acquisition rate of 10 spectra·s-1. The individual analytical fingerprints obtained were deconvoluted using MassHunter Unknown Analysis version B.07.00.
Ion Mode:POSITIVE
  
MS ID:MS002060
Analysis ID:AN002214
Instrument Name:Agilent 6224 TOF
Instrument Type:TOF
MS Type:ESI
MS Comments:The optimized MS parameters were: fragmentor 125 V, Skimmer 65 V, octopole 750 V, nebulizer pressure 10 psi, drying gas temperature at 200 °C and flow rate 10.0 L·min-1. The capillary voltage was 3500 V. Data were acquired in positive electrospray ionization (ESI) mode with a full scan from m/z 50 to 1000 at a rate of 1 spectra·s-1. The resulting CE-TOF/MS data files were cleaned of background noise and unrelated ions by the Batch Recursive Feature Extraction tool with Agilent MassHunter Profinder version B.06.00 software. Data were extracted using a data mining algorithm based on the software.
Ion Mode:POSITIVE
Capillary Voltage:3.5 kV
Dry Gas Flow:10 L·min-1
Octpole Voltage:750 V
  logo