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MB Sample ID: SA090256
| Local Sample ID: | nHTP_5neg_T21_2-21-5EY4 |
| Subject ID: | SU001309 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 0.5 - 76.5 |
| Gender: | Male and female |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001309 |
| 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 ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| nHTP_5neg_T21_2-21-5EY4 | SA090256 | FL013068 | T21 | Cohort |
Collection:
| Collection ID: | CO001303 |
| 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: | TR001324 |
| Treatment Summary: | N/A |
Sample Preparation:
| Sampleprep ID: | SP001317 |
| Sampleprep Summary: | For plasma analyses, a volume of 20μL of was extracted in 480μL of ice-cold methanol:acetonitrile:water (5:3:2). For CSF analyses, a volume of 20μL was extracted in 180μL and for cell-based experiments, 2 million cells were extracted in 1mL of the same ice-cold lysis solution. 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 | AN002061 | AN002062 |
|---|---|---|
| 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: | CH001499 |
| 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). 20uL of supernatant was injected for plasma and CSF samples, and 10uL of supernatant was injected for cell samples. Samples were resolved over a Kinetex C18 column (2.1x150 mm, 1.7μm; Phenomenex, Torrance, CA, USA) at 45°C using a 5-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: | 5MMpos_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 |
| Analytical Time: | 5min |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH001500 |
| 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). 20uL of supernatant was injected for each sample. Samples were resolved over a Kinetex C18 column (2.1x150 mm, 1.7μm; Phenomenex, Torrance, CA, USA) at 45°C using a 5-minute gradient at 450μL/minute from 0-100% B (A: 95% water/5% acetonitrile, 1mM NH4OAc; B: 95% acetonitrile/5% water, 1mM 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: | 5MMneg_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 |
| Analytical Time: | 5min |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001912 |
| Analysis ID: | AN002061 |
| 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: | MS001913 |
| Analysis ID: | AN002062 |
| 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 |
