Return to study ST001407 main page
MB Sample ID: SA114202
Local Sample ID: | q3June2014_1b |
Subject ID: | SU001481 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Age Or Age Range: | 11-20 years |
Gender: | Male and female |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU001481 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Age Or Age Range: | 11-20 years |
Gender: | Male and female |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
q3June2014_1b | SA114202 | FL014403 | Q-Standard plasma pool | Description |
Collection:
Collection ID: | CO001476 |
Collection Summary: | Eleven adolescents 12–20 years of age undergoing bariatric surgery at Cincinnati Children’s Hospital between 2006 and 2012 were offered enrollment in a prospective biospecimen repository protocol (Pediatric Obesity Tissue Repository [POTR]). Sample recruitment and other POTR features have been reported previously (Davidson et al. 2017). Intraoperatively, visceral adipose tissue (vAT) samples from the omentum, abdominal subcutaneous AT (sAT), and liver samples were obtained by the surgeon and processed immediately in an area adjacent to the operating room. All samples were snap-frozen in liquid nitrogen, then stored at −80°C. Plasma was collected pre-operatively after overnight fasting and stored at -80°C. Written informed consent was obtained from participants equal to or above 18 years old or from the parent or guardian if participants were less than 18 years old. The study was approved by the Institutional Review Board at Cincinnati Children’s Hospital. |
Sample Type: | Blood (plasma) |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR001496 |
Treatment Summary: | The objective of the observational study was to evaluate the relationship between adipose and liver tissue POPs and the plasma metabolome. All participants underwent bariatric surgery at the time of tissue collection. No other treatment or intervention was evaluated. |
Sample Preparation:
Sampleprep ID: | SP001489 |
Sampleprep Summary: | Samples are prepared for metabolomics analysis using established methods (Johnson et al. (2010). Analyst; Go et al. (2015). Tox Sci). Prior to analysis, plasma aliquots were removed from storage at -80°C and thawed on ice. Each cryotube is then vortexed briefly to ensure homogeneity, and 50 μL transferred to a clean microfuge tube. Immediately after, the plasma is treated with 100 μL of ice-cold LC-MS grade acetonitrile (Sigma Aldrich) containing 2.5 μL of internal standard solution with eight stable isotopic chemicals selected to cover a range of chemical properties. Following addition of acetonitrile, plasma is then equilibrated for 30 min on ice, upon which precipitated proteins are removed by centrifuge (16.1 ×g at 4°C for 10 min). The resulting supernatant (100 μL) is removed, added to a low volume autosampler vial and maintained at 4°C until analysis (<22 h). |
Sampleprep Protocol ID: | EmoryUniversity_HRM_SP_082016_01.pdf |
Sampleprep Protocol Filename: | EmoryUniversity_HRM_SP_082016_01.pdf |
Processing Storage Conditions: | Room temperature |
Combined analysis:
Analysis ID | AN002350 | AN002351 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | HILIC | Reversed phase |
Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
Column | Waters XBridge BEH Amide XP HILIC (50 x 2.1mm,2.5um) | Higgins endcapped C18 stainless steel (50 x 2.1mm,3um) |
MS Type | ESI | ESI |
MS instrument type | Orbitrap | Orbitrap |
MS instrument name | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap |
Ion Mode | POSITIVE | NEGATIVE |
Units | Peak intensity | Peak intensity |
Chromatography:
Chromatography ID: | CH001722 |
Chromatography Summary: | The HILIC column is operated parallel to reverse phase column for simultaneous analytical separation and column flushing through the use of a dual head HPLC pump equipped with 10- port and 6-port switching valves. During operation of HILIC separation method, the MS is operated in positive ion mode and 10 μL of sample is injected onto the HILIC column while the reverse phase column is flushing with wash solution. Flow rate is maintained at 0.35 mL/min until 1.5 min, increased to 0.4 mL/min at 4 min and held for 1 min. Solvent A is 100% LC-MS grade water, solvent B is 100% LC-MS grade acetonitrile and solvent C is 2% formic acid (v/v) in LC-MS grade water. Initial mobile phase conditions are 22.5% A, 75% B, 2.5% C hold for 1.5 min, with linear gradient to 77.5% A, 20% B, 2.5% C at 4 min, hold for 1 min, resulting in a total analytical run time of 5 min. During the flushing phase (reverse phase analytical separation), the HILIC column is equilibrated with a wash solution of 77.5% A, 20% B, 2.5% C. |
Instrument Name: | Thermo Dionex Ultimate 3000 |
Column Name: | Waters XBridge BEH Amide XP HILIC (50 x 2.1mm,2.5um) |
Column Temperature: | 60 |
Flow Gradient: | Initial mobile phase conditions are 22.5% A, 75% B, 2.5% C hold for 1.5 min, with linear gradient to 77.5% A, 20% B, 2.5% C at 4 min, hold for 1 min, resulting in a total analytical run time of 5 min. |
Flow Rate: | 0.35- 0.4 mL/min |
Internal Standard: | [13C6]-D-glucose, [15N,13C5]- L-methionine, [13C5]-L-glutamic acid, [15N]-L-tyrosine, [3,3-13C2]-cystine, [trimethyl- 13C3]-caffeine, [U-13C5, U-15N2]-L-glutamine |
Sample Injection: | 10 uL |
Solvent A: | 100% water(A), 100% acetonitrile(B), 100% water; 2% formic acid(C) |
Solvent B: | 100% water(A), 100% acetonitrile(B), 100% water; 2% formic acid(C) |
Analytical Time: | 5 min |
Chromatography Type: | HILIC |
Chromatography ID: | CH001723 |
Chromatography Summary: | The C18 column is operated parallel to the HILIC column for simultaneous analytical separation and column flushing through the use of a dual head HPLC pump equipped with 10-port and 6- port switching valves. During operation of the C18 method, the MS is operated in negative ion mode and 10 μL of sample is injected onto the C18 column while the HILIC column is flushing with wash solution. Flow rate is maintained at 0.4 mL/min until 1.5 min, increased to 0.5 mL/min at 2 min and held for 3 min. Solvent A is 100% LC-MS grade water, solvent B is 100% LC-MS grade acetonitrile and solvent C is 10mM ammonium acetate in LC-MS grade water. Initial mobile phase conditions are 60% A, 35% B, 5% C hold for 0.5 min, with linear gradient to 0% A, 95% B, 5% C at 1.5 min, hold for 3.5 min, resulting in a total analytical run time of 5 min. During the flushing phase (HILIC analytical separation), the C18 column is equilibrated with a wash solution of 0% A, 95% B, 5% C until 2.5 min, followed by an equilibration solution of 60% A, 35% B, 5% C for 2.5 min. |
Instrument Name: | Thermo Dionex Ultimate 3000 |
Column Name: | Higgins endcapped C18 stainless steel (50 x 2.1mm,3um) |
Column Temperature: | 60 |
Flow Gradient: | Initial mobile phase conditions are 60% A, 35% B, 5% C hold for 0.5 min, with linear gradient to 0% A, 95% B, 5% C at 1.5 min, hold for 3.5 min, resulting in a total analytical run time of 5 min. |
Flow Rate: | Flow rate is maintained at 0.4 mL/min until 1.5 min, increased to 0.5 mL/min at 2 min and held for 3 min. |
Internal Standard: | [13C6]-D-glucose, [15N,13C5]- L-methionine, [13C5]-L-glutamic acid, [15N]-L-tyrosine, [3,3-13C2]-cystine, [trimethyl- 13C3]-caffeine, [U-13C5, U-15N2]-L-glutamine |
Sample Injection: | 10 uL |
Solvent A: | 100% water(A), 100% acetonitrile(B), 100%water; 10mM ammonium acetate(C) |
Solvent B: | 100% water(A), 100% acetonitrile(B), 100%water; 10mM ammonium acetate(C) |
Analytical Time: | 5 min |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS002192 |
Analysis ID: | AN002350 |
Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The high-resolution mass spectrometer was operated at 120,000 resolution and mass-to-charge ratio (m/z) range 85-1275. Probe temperature, capillary temperature, sweep gas and S-Lens RF levels were maintained at 200°C, 300°C, 1 arbitrary units (AU), and 45, respectively. Additional source settings were optimized for sensitivity using a standard mixture, tune settings for sheath gas, auxiliary gas, sweep gas and spray voltage setting were 45 AU, 25 AU and 3.5 kV, respectively. Maximum C-trap injection times were set at 100 milliseconds and automatic gain control target 1 × 106. During untargeted data acquisition, no exclusion or inclusion masses were selected, and data was acquired in MS1 mode only. Raw data files were then extracted using apLCMS (Yu et al. 2009) at five different peak detection settings that have been separately optimized for detection of a wide range of peak intensities and abundances. Peaks detected during each injection were aligned using a mass tolerance of 5 ppm (parts-per-million) and retention grouping was accomplished using non-parametric density estimation grouping, with a maximum retention time deviation of 30 seconds. The resulting feature tables were merged using xMSanalyzer, which identifies overlapping or unique features detected across the different peak detection parameters, and retains the peak with the lowest replicate CV and non-detects for inclusion in the final feature table (Uppal et al. 2013). All R-scripts for data extraction with apLCMS and data merging with xMSanalyzer are provided in the supplementary material. Uniquely detected ions consisted of m/z, retention time and ion abundance, referred to as m/z features. Prior to data analysis, triplicate m/z features averaged and filtered to remove those with triplicate coefficient of variation (CV) ≥ 100% and non-detected values greater than 10%. |
Ion Mode: | POSITIVE |
Capillary Temperature: | 300C |
Ion Source Temperature: | 200C |
Ion Spray Voltage: | 3.5kV |
Ionization: | Postive |
Mass Accuracy: | 5ppm |
Source Temperature: | 200C |
MS ID: | MS002193 |
Analysis ID: | AN002351 |
Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The high-resolution mass spectrometer was operated at 120,000 resolution and mass-to-charge ratio (m/z) range 85-1275. Probe temperature, capillary temperature, sweep gas and S-Lens RF levels were maintained at 200°C, 300°C, 1 arbitrary units (AU), and 45, respectively. Additional source settings were optimized for sensitivity using a standard mixture, tune settings for sheath gas, auxiliary gas, sweep gas and spray voltage setting were 45 AU, 25 AU and 3.5 kV, respectively. Maximum C-trap injection times were set at 100 milliseconds and automatic gain control target 1 × 106. During untargeted data acquisition, no exclusion or inclusion masses were selected, and data was acquired in MS1 mode only. Raw data files were then extracted using apLCMS (Yu et al. 2009) at five different peak detection settings that have been separately optimized for detection of a wide range of peak intensities and abundances. Peaks detected during each injection were aligned using a mass tolerance of 5 ppm (parts-per-million) and retention grouping was accomplished using non-parametric density estimation grouping, with a maximum retention time deviation of 30 seconds. The resulting feature tables were merged using xMSanalyzer, which identifies overlapping or unique features detected across the different peak detection parameters, and retains the peak with the lowest replicate CV and non-detects for inclusion in the final feature table (Uppal et al. 2013). All R-scripts for data extraction with apLCMS and data merging with xMSanalyzer are provided in the supplementary material. Uniquely detected ions consisted of m/z, retention time and ion abundance, referred to as m/z features. Prior to data analysis, triplicate m/z features averaged and filtered to remove those with triplicate coefficient of variation (CV) ≥ 100% and non-detected values greater than 10%. |
Ion Mode: | NEGATIVE |
Capillary Temperature: | 300C |
Ion Source Temperature: | 200C |
Ion Spray Voltage: | 3.5kV |
Ionization: | NEgative |
Mass Accuracy: | 5ppm |
Source Temperature: | 200C |