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MB Sample ID: SA198304
| Local Sample ID: | 20190125 CarT_Prodh2_KI_Vector3 M105 |
| Subject ID: | SU002168 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002168 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 20190125 CarT_Prodh2_KI_Vector3 M105 | SA198304 | FL024100 | PRODH2(Stop)-CAR | Genotype |
| 20190125 CarT_Prodh2_KI_Vector3 M105 | SA198304 | FL024100 | PRODH2(Stop) | Treatment |
Collection:
| Collection ID: | CO002161 |
| Collection Summary: | cell were counted, washed twice with PBS then used for metabolite extraction. |
| Sample Type: | T-cells |
Treatment:
| Treatment ID: | TR002180 |
| Treatment Summary: | T cells were electroporated with Cas9-RNP, then transduced with CD22-CAR;PRODH2 or PRODH2(Stop) AAV, CAR-T cells were collected for metabolite extraction. |
Sample Preparation:
| Sampleprep ID: | SP002174 |
| Sampleprep Summary: | After normalizing cell counts, 800 μL of 80 % (vol / vol) HPLC-grade methanol (Sigma) (precooled to -80 oC on dry ice) was added to fresh cells in a 1.5-mL microcentrifuge tube, then tubes were put on dry ice for 30 minutes. The tubes were then incubated on ice for 20 minutes and centrifuged at 15,000 x g for 15 min at 4 oC to pellet the cell debris. The metabolite-containing supernatant was transferred to a new 1.5-mL microcentrifuge tube on dry ice. Metabolite extraction was repeated with 400 μL of 80 % (vol / vol) HPLC-grade methanol. The cell lysate / methanol mixtures were dried by Speedvac at room temperature. The metabolites were dissolved again with 80 % (vol / vol) methanol, then centrifuged at 18,000 x g for 10 min to remove any particulates, and the metabolite mixtures were stored at -80 oC until LC-MS analysis. |
Combined analysis:
| Analysis ID | AN003401 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Agilent 6490 |
| Column | Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | Agilent 6490 QQQ |
| Ion Mode | POSITIVE |
| Units | ppm |
Chromatography:
| Chromatography ID: | CH002514 |
| Instrument Name: | Agilent 6490 |
| Column Name: | Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003168 |
| Analysis ID: | AN003401 |
| Instrument Name: | Agilent 6490 QQQ |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Two metabolomics strategies were adopted, i.e. untargeted metabolomics (aiming to unbiasedly detect all detectable metabolites) and targeted approaches (aiming to detect specifically defined metabolites, such as related metabolites in the proline metabolism and T cell metabolism). For untargeted metabolomics analysis, the optimized workflow consists of automated peak detection and integration, peak alignment, background noise subtraction, and multivariate data analysis. These steps were carried out for comprehensive metabolite phenotyping of the two groups using Agilent Mass Hunter Qualitative Analysis Software (Version B.07.0.0, build 7.0.7024.0) and Agilent Mass Profiler Professional (Version 14.5-Build 2772). The metabolites were first putatively identified based on accurate mass match (accurate mass ± 30 ppm error) and fragmentation pattern match. Putative structural annotation was carried out by searching the metabolite databases HMDB (http://www.hmdb.ca/) and METLIN (http:// metlin.scripps.edu) using the mass-to-charge ratio of the metabolic features. |
| Ion Mode: | POSITIVE |
