Summary of Study ST003551
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 PR002187. The data can be accessed directly via it's Project DOI: 10.21228/M8XN8V 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 | ST003551 |
| Study Title | Metabolomics analysis of N-methyl-arginine-treated HEK293 control (sgTomato) and ALDH7A1-deficient (sgALDH7A1) cells. |
| Study Summary | Metabolite analysis of HEK293 cells treated with N-methyl-arginine (NMA), an inhibitor of lysine/arginine transport. Cells were treated for 20-24 hours with varying concentration of NMA, briefly washed with ice cold 0.9% saline, and quenched with 1 mL of ice cold 80% methanol containing 1 ul of stable-isotope-labeled internal amino acid standard mix. Metabolites were concentrated using a SpeedVac until dry and analyzed by LCMS. The LC was coupled to an Exploris 240 mass spectrometer operated in a polarity switching data-dependent Top 5 mode. Full MS scan parameters for both positive and negative mode were set to 67-1000 m/z at a resolution of 120k and ddMS2 were collected at a resolution of 30k. |
| 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: | PR002187 |
| Project DOI: | doi: 10.21228/M8XN8V |
| Project Title: | N-methyl-arginine significantly reduces intracellular levels of cationic amino acids and related catabolites in a dose-dependent manner. |
| Project Type: | Manuscript |
| Project Summary: | N-methyl-arginine inhibits the uptake and metabolism of lysine and arginine by competing for similar transporters. In this project, we aimed to determine the cellular effects of N-methyl-arginine using metabolomics by treating HEK293 control (sgTom) or ALDH7A1-KO (sgALDH7A1) cells for 20-24 hours. N-methyl-arginine treatment significantly reduced the levels of arginine, lysine, saccharopine, aminoadipate, piperideine 6-carboxylate, and pipecolate in a dose-dependent manner. |
| 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: | SU003680 |
| Subject Type: | Mammal |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Not applicable |
| Species Group: | Mammals |
Factors:
Subject type: Mammal; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Genotype | Treatment |
|---|---|---|---|---|
| SA387888 | HEK293_TOM_10NMA_PM3 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | N-methyl-arginine (10 mM) |
| SA387889 | HEK293_TOM_10NMA_PM2 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | N-methyl-arginine (10 mM) |
| SA387890 | HEK293_TOM_10NMA_PM1 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | N-methyl-arginine (10 mM) |
| SA387891 | HEK293_TOM_20NMA_PM3 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | N-methyl-arginine (20 mM) |
| SA387892 | HEK293_TOM_20NMA_PM2 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | N-methyl-arginine (20 mM) |
| SA387893 | HEK293_TOM_20NMA_PM1 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | N-methyl-arginine (20 mM) |
| SA387894 | HEK293_TOM_5NMA_PM1 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | N-methyl-arginine (5 mM) |
| SA387895 | Tom_NMA_PM1 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | N-methyl-arginine (5 mM) |
| SA387896 | Tom_NMA_PM2 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | N-methyl-arginine (5 mM) |
| SA387897 | Tom_NMA_PM3 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | N-methyl-arginine (5 mM) |
| SA387898 | Tom_control_PM2 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | PBS |
| SA387899 | Tom_control_PM1 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | PBS |
| SA387900 | HEK293_TOM_0NMA_PM3 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | PBS |
| SA387901 | HEK293_TOM_0NMA_PM1 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | PBS |
| SA387902 | HEK293_TOM_0NMA_PM2 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | PBS |
| SA387903 | Tom_control_PM3 | Cultured cell line | CRISPR/Cas9 Control (sgTomato) | PBS |
| SA387904 | HEK293_KO_10NMA_PM2 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | N-methyl-arginine (10 mM) |
| SA387905 | HEK293_KO_10NMA_PM3 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | N-methyl-arginine (10 mM) |
| SA387906 | HEK293_KO_10NMA_PM1 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | N-methyl-arginine (10 mM) |
| SA387907 | HEK293_KO_20NMA_PM3 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | N-methyl-arginine (20 mM) |
| SA387908 | HEK293_KO_20NMA_PM2 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | N-methyl-arginine (20 mM) |
| SA387909 | HEK293_KO_20NMA_PM1 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | N-methyl-arginine (20 mM) |
| SA387910 | HEK293_KO_5NMA_PM1 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | N-methyl-arginine (5 mM) |
| SA387911 | KO_NMA_PM3 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | N-methyl-arginine (5 mM) |
| SA387912 | KO_NMA_PM1 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | N-methyl-arginine (5 mM) |
| SA387913 | KO_control_PM2 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | PBS |
| SA387914 | KO_control_PM1 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | PBS |
| SA387915 | KO_control_PM3 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | PBS |
| SA387916 | HEK293_KO_0NMA_PM3 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | PBS |
| SA387917 | HEK293_KO_0NMA_PM1 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | PBS |
| SA387918 | HEK293_KO_0NMA_PM2 | Cultured cell line | CRISPR/Cas9 KO (sgALDH7A1) | PBS |
| Showing results 1 to 31 of 31 |
Collection:
| Collection ID: | CO003673 |
| Collection Summary: | Cells were plated in DMEM containing 10% FBS and allowed to attach overnight. The following morning, the media was aspirated and replaced with DMEM containing 10% dialyzed FBS and a vehicle (PBS) or 5-20 mM of N-methyl-arginine. Cells were incubated overnight between 20-24 hours and metabolites were extracted for analysis by LCMS. |
| Sample Type: | Epithelial cells |
Treatment:
| Treatment ID: | TR003689 |
| Treatment Summary: | Cells were treated with a vehicle (PBS) or between 5-20 mM of N-methyl-arginine added to DMEM containing 10% dialyzed FBS for 20-24 hours before metabolite extraction and analysis by LCMS. |
| Treatment Compound: | N-methyl-arginine |
| Treatment Dose: | 5-20 mM |
| Treatment Vehicle: | PBS |
Sample Preparation:
| Sampleprep ID: | SP003687 |
| 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 | AN005837 | AN005838 |
|---|---|---|
| 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 Orbitrap Exploris 240 | Thermo Orbitrap Exploris 240 |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | Ion counts | Ion counts |
Chromatography:
| Chromatography ID: | CH004434 |
| 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: | MS005557 |
| Analysis ID: | AN005837 |
| Instrument Name: | Thermo Orbitrap 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: | MS005558 |
| Analysis ID: | AN005838 |
| Instrument Name: | Thermo Orbitrap 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 |
