Summary of Study ST003550
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002186. The data can be accessed directly via it's Project DOI: 10.21228/M82B9C This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST003550 |
| Study Title | Stable-isotope lysine tracing in primary mouse astrocytes and HEK293 cells. |
| Study Summary | Stable-isotope tracing using unlabeled, 13C6-, alpha-15N-, or epsilon-15N- labeled L-lysine in primary mouse astrocytes and HEK293 cells. Primary mouse astrocytes were isolated from wild-type C57BL/6J mice or Aldh7a1+/- (Het) or Aldh7a1-/- (Hom) mice and cultured for 24-hours in DMEM containing one of the above tracers and 10% dialyzed FBS. Parental, sgTomato, or sgALDH7A1 HEK293 cells were cultured for 24-hours in DMEM containing one of the above tracers and 10% dialyzed FBS. Metabolites were extracted and analyzed by LCMS. |
| Institute | University of British Columbia |
| Department | Biochemistry & Molecular Biology |
| Laboratory | Parker laboratory |
| Last Name | Parker |
| First Name | Seth |
| Address | 950 W 28th Ave, Vancouver, British Columbia, V6H 0B3, Canada |
| seth.parker@bcchr.ca | |
| Phone | 6048753121 |
| Submit Date | 2024-11-01 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-11-15 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002186 |
| Project DOI: | doi: 10.21228/M82B9C |
| Project Title: | Stable-isotope lysine tracing to differentiate utilization of the saccharopine and/or pipecolate pathways in HEK293 cells and primary mouse astrocytes. |
| Project Type: | Manuscript |
| Project Summary: | L-lysine (Lysine) is an essential proteinogenic amino acid that is acquired from a protein-rich diet. Lysine is metabolized to fuel acyl-CoA and L-carnitine synthesis in cells and tissues. The two main catabolic pathways, named after their products pipecolate or saccharopine, differ in their cytosolic or mitochondrial localization, respectively, but converge at the mitochondrial enzyme α-aminoadipic semialdehyde dehydrogenase (ALDH7A1, or antiquitin). To differentiate between the utilization of these two pathways, we cultured HEK293 cells or primary mouse astrocytes with unlabeled L-lysine, 13C6-L-lysine, alpha-15N-L-lysine, or epsilon-15N-L-lysine for 24 hours and analyzed atom incorporation into downstream metabolites using LCMS. Our results suggest that both cell types exclusively use the saccharopine pathway. In addition, we show that pipecolate can be generated from interconversion of piperideine 6-carboxylate rather than from activity of the pipecolate pathway, which was silent in both cell types. |
| Institute: | University of British Columbia |
| Department: | Biochemistry & Molecular Biology |
| Laboratory: | Parker laboratory |
| Last Name: | Parker |
| First Name: | Seth |
| Address: | 950 W 28th Ave, Vancouver, British Columbia, V6H 0B3, Canada |
| Email: | seth.parker@bcchr.ca |
| Phone: | 6048753121 |
Subject:
| Subject ID: | SU003679 |
| Subject Type: | Cultured cells |
| Subject Species: | Mouse and Human |
| Gender: | Male and female |
Factors:
Subject type: Cultured cells; Subject species: Mouse and Human (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Isotope Tracer |
|---|---|---|---|
| SA387828 | HEK293_13C6Lys_3 | HEK293 | 13C6-L-lysine |
| SA387829 | HEK293_13C6Lys_2 | HEK293 | 13C6-L-lysine |
| SA387830 | HEK293_13C6Lys_1 | HEK293 | 13C6-L-lysine |
| SA387831 | HEK293_sgTom_a15NLys_1 | HEK293 | alpha-15N-L-lysine |
| SA387832 | HEK293_a15NLys_2 | HEK293 | alpha-15N-L-lysine |
| SA387833 | HEK293_a15NLys_1 | HEK293 | alpha-15N-L-lysine |
| SA387834 | HEK293_a15NLys_3 | HEK293 | alpha-15N-L-lysine |
| SA387835 | HEK293_sgTom_a15NLys_2 | HEK293 | alpha-15N-L-lysine |
| SA387836 | HEK293_sgTom_a15NLys_3 | HEK293 | alpha-15N-L-lysine |
| SA387837 | HEK293_sgALDH7A1_a15NLys_1 | HEK293 | alpha-15N-L-lysine |
| SA387838 | HEK293_sgALDH7A1_a15NLys_2 | HEK293 | alpha-15N-L-lysine |
| SA387839 | HEK293_sgALDH7A1_a15NLys_3 | HEK293 | alpha-15N-L-lysine |
| SA387840 | HEK293_e15NLys_1 | HEK293 | epsilon-15N-L-lysine |
| SA387841 | HEK293_e15NLys_3 | HEK293 | epsilon-15N-L-lysine |
| SA387842 | HEK293_e15NLys_2 | HEK293 | epsilon-15N-L-lysine |
| SA387843 | HEK293_sgTom_unlabel_2 | HEK293 | unlabeled L-lysine |
| SA387844 | HEK293_sgTom_unlabel_1 | HEK293 | unlabeled L-lysine |
| SA387845 | HEK293_sgALDH7A1_unlabel_2 | HEK293 | unlabeled L-lysine |
| SA387846 | HEK293_sgALDH7A1_unlabel_3 | HEK293 | unlabeled L-lysine |
| SA387847 | HEK293_sgTom_unlabel_3 | HEK293 | unlabeled L-lysine |
| SA387848 | HEK293_sgALDH7A1_unlabel_1 | HEK293 | unlabeled L-lysine |
| SA387849 | HEK293_unlabeled_3 | HEK293 | unlabeled L-lysine |
| SA387850 | HEK293_unlabeled_1 | HEK293 | unlabeled L-lysine |
| SA387851 | HEK293_unlabeled_2 | HEK293 | unlabeled L-lysine |
| SA387852 | Hom_C6_3 | Primary mouse astrocytes | 13C6-L-lysine |
| SA387853 | Hom_C6_2 | Primary mouse astrocytes | 13C6-L-lysine |
| SA387854 | Hom_C6_1 | Primary mouse astrocytes | 13C6-L-lysine |
| SA387855 | Het_C6_3 | Primary mouse astrocytes | 13C6-L-lysine |
| SA387856 | Astrocytes_13C6Lys_SP3 | Primary mouse astrocytes | 13C6-L-lysine |
| SA387857 | Astrocytes_13C6Lys_SP2 | Primary mouse astrocytes | 13C6-L-lysine |
| SA387858 | Astrocytes_13C6Lys_SP1 | Primary mouse astrocytes | 13C6-L-lysine |
| SA387859 | Het_C6_2 | Primary mouse astrocytes | 13C6-L-lysine |
| SA387860 | Het_C6_1_rerun2 | Primary mouse astrocytes | 13C6-L-lysine |
| SA387861 | Het_Na_2 | Primary mouse astrocytes | alpha-15N-L-lysine |
| SA387862 | Hom_Na_3 | Primary mouse astrocytes | alpha-15N-L-lysine |
| SA387863 | Hom_Na_2 | Primary mouse astrocytes | alpha-15N-L-lysine |
| SA387864 | Hom_Na_1 | Primary mouse astrocytes | alpha-15N-L-lysine |
| SA387865 | Het_Na_3 | Primary mouse astrocytes | alpha-15N-L-lysine |
| SA387866 | Het_Na_1 | Primary mouse astrocytes | alpha-15N-L-lysine |
| SA387867 | Astrocytes_a15NLys_SP2 | Primary mouse astrocytes | alpha-15N-L-lysine |
| SA387868 | Astrocytes_a15NLys_SP1 | Primary mouse astrocytes | alpha-15N-L-lysine |
| SA387869 | Astrocytes_a15NLys_SP3 | Primary mouse astrocytes | alpha-15N-L-lysine |
| SA387870 | Astrocytes_e15NLys_SP3 | Primary mouse astrocytes | epsilon-15N-L-lysine |
| SA387871 | Het_Ne_1 | Primary mouse astrocytes | epsilon-15N-L-lysine |
| SA387872 | Het_Ne2 | Primary mouse astrocytes | epsilon-15N-L-lysine |
| SA387873 | Het_Ne_3 | Primary mouse astrocytes | epsilon-15N-L-lysine |
| SA387874 | Astrocytes_e15NLys_SP2 | Primary mouse astrocytes | epsilon-15N-L-lysine |
| SA387875 | Astrocytes_e15NLys_SP1 | Primary mouse astrocytes | epsilon-15N-L-lysine |
| SA387876 | Hom_Ne3 | Primary mouse astrocytes | epsilon-15N-L-lysine |
| SA387877 | Hom_Ne2 | Primary mouse astrocytes | epsilon-15N-L-lysine |
| SA387878 | Hom_Ne1 | Primary mouse astrocytes | epsilon-15N-L-lysine |
| SA387879 | Hom_Unlabelled_3 | Primary mouse astrocytes | unlabeled L-lysine |
| SA387880 | Astrocytes_unlabeled_SP2 | Primary mouse astrocytes | unlabeled L-lysine |
| SA387881 | Het_Unlabelled_3 | Primary mouse astrocytes | unlabeled L-lysine |
| SA387882 | Hom_Unlabelled_2 | Primary mouse astrocytes | unlabeled L-lysine |
| SA387883 | Hom_Unlabelled_1 | Primary mouse astrocytes | unlabeled L-lysine |
| SA387884 | Astrocytes_unlabeled_SP3 | Primary mouse astrocytes | unlabeled L-lysine |
| SA387885 | Het_Unlabelled_1 | Primary mouse astrocytes | unlabeled L-lysine |
| SA387886 | Het_Unlabelled_2 | Primary mouse astrocytes | unlabeled L-lysine |
| SA387887 | Astrocytes_unlabeled_SP1 | Primary mouse astrocytes | unlabeled L-lysine |
| Showing results 1 to 60 of 60 |
Collection:
| Collection ID: | CO003672 |
| Collection Summary: | Cultured cells were rinsed with ice cold 0.9% saline and metabolites were extracted using ice cold 80% methanol and dried using a SpeedVac before LCMS analysis. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003688 |
| Treatment Summary: | DMEM containing 10% dialyzed FBS and 0.8 mM of either unlabeled L-lysine, 13C6-L-lysine, alpha-15N-L-lysine, or epsilon-15N-L-lysine. Cells were cultured in tracing media for 24 hours before metabolite extraction and analysis by LCMS. |
| Treatment Compound: | Stable-isotope lysine tracers |
| Treatment Dose: | 0.8 mM (Normal DMEM concentration) |
| Cell Media: | DMEM |
Sample Preparation:
| Sampleprep ID: | SP003686 |
| Sampleprep Summary: | Dried samples were reconstituted in 50 µL of HPLC-grade water. Samples were vortexed for ~10 minutes, then centrifuged at 21,000 x g for 15 min at 4°C. 40 microliters were transferred to LC vials containing glass inserts for analysis. |
Combined analysis:
| Analysis ID | AN005835 | AN005836 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Vanquish | Thermo Vanquish |
| Column | Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) | Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Exploris 240 | Thermo Exploris 240 |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Ion counts | Ion counts |
Chromatography:
| Chromatography ID: | CH004433 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 25°C |
| Flow Gradient: | 80-20%B (0-30 min), 20-20%B (30-40 minute), and 20-80%B (40-40.5 minute); the LC column was re-equilibrated using 80-80%B from 40.5-52 minute before subsequent injections |
| Flow Rate: | 100 µL/min |
| Solvent A: | 100% Water; 10 mM Ammonium Carbonate, pH 9.0 |
| Solvent B: | 100% Acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS005555 |
| Analysis ID: | AN005835 |
| Instrument Name: | Thermo Exploris 240 |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The LC was coupled to a Thermo Scientific Exploris 240 mass spectrometer operating in heated electrospray ionization mode (HESI) for analysis. The following parameters were set for HESI: spray voltage 3.4 kV (positive) and 2 kV (negative), static spray voltage, sheath gas 25, aux gas 5, sweep gas 0.5, ion transfer tube temperature 320°C, and vaporizer temperature 75°C. The global parameters included an expected peak width of 20 seconds, mild trapping, and a default charge state of 1. A 40-min polarity switching data-dependent Top 5 method was used for positive mode and a data-dependent Top 3 method was used for negative mode. Full MS scan parameters for both positive and negative modes were set as follows: scan range 67-1000 m/z collected in profile mode, Orbitrap resolution 120,000, RF lens 70%, AGC target of 300%, and maximum injection time set to automatic. ddMS2 for positive mode were collected in centroid mode at an Orbitrap resolution of 30,000, isolation window of 1.5 m/z, an AGC target set to standard, a maximum injection time set to automatic, and a normalized collision energy set to 10%, 30%, and 80%. ddMS2 for negative mode were collected in centroid mode at an Orbitrap resolution of 30,000, isolation window of 2 m/z, an AGC target set to standard, a maximum injection time set to automatic, and a normalized collision energy set to 30%. For both positive and negative ddMS2, we applied an intensity threshold of 5e4 and a dynamic exclusion of 5 ppm for 10 seconds, excluding isotopes. A targeted selected ion monitoring (tSIM) scan was also included for pipecolate and P6C/P2C, and the retention time ranges were based on elution of authentic standards (pipecolate) or from positive samples (Aldh7a1-deficient tissues). The tSIM scan for pipecolate was collected from 8-12 minutes in negative mode at an isolation window of 4 m/z (for metabolomics) or 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, and collected in profile mode. The tSIM scan for P6C/P2C was collected from 6-10 minutes in positive mode at an isolation window of 4 m/z (for metabolomics) or 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, and collected in profile mode. |
| Ion Mode: | POSITIVE |
| MS ID: | MS005556 |
| Analysis ID: | AN005836 |
| Instrument Name: | Thermo Exploris 240 |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The LC was coupled to a Thermo Scientific Exploris 240 mass spectrometer operating in heated electrospray ionization mode (HESI) for analysis. The following parameters were set for HESI: spray voltage 3.4 kV (positive) and 2 kV (negative), static spray voltage, sheath gas 25, aux gas 5, sweep gas 0.5, ion transfer tube temperature 320°C, and vaporizer temperature 75°C. The global parameters included an expected peak width of 20 seconds, mild trapping, and a default charge state of 1. A 40-min polarity switching data-dependent Top 5 method was used for positive mode and a data-dependent Top 3 method was used for negative mode. Full MS scan parameters for both positive and negative modes were set as follows: scan range 67-1000 m/z collected in profile mode, Orbitrap resolution 120,000, RF lens 70%, AGC target of 300%, and maximum injection time set to automatic. ddMS2 for positive mode were collected in centroid mode at an Orbitrap resolution of 30,000, isolation window of 1.5 m/z, an AGC target set to standard, a maximum injection time set to automatic, and a normalized collision energy set to 10%, 30%, and 80%. ddMS2 for negative mode were collected in centroid mode at an Orbitrap resolution of 30,000, isolation window of 2 m/z, an AGC target set to standard, a maximum injection time set to automatic, and a normalized collision energy set to 30%. For both positive and negative ddMS2, we applied an intensity threshold of 5e4 and a dynamic exclusion of 5 ppm for 10 seconds, excluding isotopes. A targeted selected ion monitoring (tSIM) scan was also included for pipecolate and P6C/P2C, and the retention time ranges were based on elution of authentic standards (pipecolate) or from positive samples (Aldh7a1-deficient tissues). The tSIM scan for pipecolate was collected from 8-12 minutes in negative mode at an isolation window of 4 m/z (for metabolomics) or 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, and collected in profile mode. The tSIM scan for P6C/P2C was collected from 6-10 minutes in positive mode at an isolation window of 4 m/z (for metabolomics) or 18 m/z (for isotope-tracing experiments, to include m/z shifts), an Orbitrap resolution of 120,000, a RF lens at 70%, an automatic maximum injection time, and collected in profile mode. |
| Ion Mode: | NEGATIVE |
