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MB Sample ID: SA090692

Local Sample ID:Nexus_T21_4neg-61
Subject ID:SU001311
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:0.5 - 76.5
Gender:Male and female

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Subject:

Subject ID:SU001311
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:0.5 - 76.5
Gender:Male and female

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
Nexus_T21_4neg-61SA090692FL013072T21Cohort

Collection:

Collection ID:CO001305
Collection Summary:All human subjects in this study were consented according to Colorado Multiple Institutional Review Board (COMIRB)-approved protocols. Written informed consent was obtained from parents or guardians of participants under the age of 18, and assent was obtained from participants over the age of 7 who were cognitively able to assent. Deidentified plasma samples for Cohort 1 were obtained from the Translational Nexus Clinical Data Registry and Biobank (University of Colorado Anschutz Medical Campus, COMIRB 08-1276). Additional plasma and WBC samples were obtained through the Crnic’s Institute Human Trisome Project (University of Colorado Anschutz Medical Campus, COMIRB 15-2170, www.trisome.org). Plasma was collected in Vacutainer tubes (EDTA–purple capped or Lithium heparin–light green capped) and stored at -80°C. Participant medical history was collected by the respective biobanks.
Sample Type:Blood (plasma)

Treatment:

Treatment ID:TR001326
Treatment Summary:N/A

Sample Preparation:

Sampleprep ID:SP001319
Sampleprep Summary:For plasma analyses, a volume of 20μL of was extracted in 480μL of ice-cold methanol:acetonitrile:water (5:3:2). Subsequently, these solutions were vortexed for 30 minutes at 4°C.Insoluble proteins were pelleted by centrifugation (10 minutes at 4°C and 12,000 g) and supernatants were collected and stored at -80°C until analysis. For quantitative analysis of kynurenine pathway (KP) metabolites, supernatants were spun in a Speedvac until dry and resuspended in 0.1% formic acid in water as previously described (PMID: 30213797, 30143553).

Combined analysis:

Analysis ID AN002065 AN002066
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish
Column Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE
Units relative abundance relative abundance

Chromatography:

Chromatography ID:CH001503
Chromatography Summary:UHPLC-MS metabolomics analyses were performed using a Vanquish UHPLC system coupled online to a Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany). Samples were resolved over a Kinetex C18 column (2.1x150 mm, 1.7μm; Phenomenex, Torrance, CA, USA) at 45°C using a 4 minute gradient at 450μL/minute from 5-95% B (A: water/0.1% formic acid; B: acetonitrile/0.1% formic acid) for positive mode. To monitor possible technical variability, aliquots of each of the individual samples were combined to make technical replicates, which were run after every 15 samples. Additionally, in each experiment, several lysis solution aliquots were run as blanks for artifact identification.
Methods Filename:4MMpos_method.pdf
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Column Temperature:45
Flow Gradient:5-95% B
Flow Rate:450uL/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH001504
Chromatography Summary:UHPLC-MS metabolomics analyses were performed using a Vanquish UHPLC system coupled online to a Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany). Samples were resolved over a Kinetex C18 column (2.1x150 mm, 1.7μm; Phenomenex, Torrance, CA, USA) at 45°C using a 4 minute gradient at 450μL/minute from 0-100% B (A: 95% water/5% acetonitrile, 5mM NH4OAc; B: 95% acetonitrile/5% water, 5mM NH4OAc) for negative mode. To monitor possible technical variability, aliquots of each of the individual samples were combined to make technical replicates, which were run after every 15 samples. Additionally, in each experiment, several lysis solution aliquots were run as blanks for artifact identification.
Methods Filename:4MMneg_method.pdf
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Column Temperature:45
Flow Gradient:0-100% B
Flow Rate:450uL/min
Solvent A:95% water/5% acetonitrile; 1 mM ammonium acetate
Solvent B:95% acetonitrile/5% water; 1 mM ammonium acetate
Chromatography Type:Reversed phase

MS:

MS ID:MS001916
Analysis ID:AN002065
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The Q Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) was operated in positive ion mode using electrospray ionization, scanning in Full MS mode (1μscan) from 65 to 975 m/z at 70,000 resolution, with 4 kV spray voltage, 45 sheath gas, 15 auxiliary gas. MS analysis and data elaboration were performed as described74,75. Calibration was performed prior to analysis using the PierceTM Positive Ion Calibration Solution (Thermo Fisher Scientific).
Ion Mode:POSITIVE
  
MS ID:MS001917
Analysis ID:AN002066
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The Q Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) was operated in negative ion mode using electrospray ionization, scanning in Full MS mode (1μscan) from 65 to 975 m/z at 70,000 resolution, with 4 kV spray voltage, 45 sheath gas, 15 auxiliary gas. MS analysis and data elaboration were performed as described74,75. Calibration was performed prior to analysis using the PierceTM Negative Ion Calibration Solution (Thermo Fisher Scientific).
Ion Mode:NEGATIVE
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