Summary of Study ST003548
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 PR002184. The data can be accessed directly via it's Project DOI: 10.21228/M89V56 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 | ST003548 |
| Study Title | Metabolomics of Aldh7a1-/- mice treated with lysine alpha-oxidase from Trichoderma viride |
| Study Summary | Metabolite analysis of brain, liver, and plasma isolated from Aldh7a1-/- (n=3/each) fed diets consisting of 0.9% w/w lysine and 18 ppm pyridoxine administered with PBS or 150 U/kg lysine alpha-oxidase (LOX) from Trichoderma viride. Tissues were cryo-homogenized using a liquid nitrogen cooled mortar and pestle into a fine powder and approximately 25-50 mg of each tissue was transferred to a pre-weighed homogenization tube to which ~20 2.3 mm zirconia beads and 1 mL of 80% ice cold methanol were added. Tissues were homogenized using a bead mill and cleared by centrifugation at 4C, where each of the 3-4 cycles consisted of a 60 second homogenization at 6 m/s followed by a 30 second pause. A volume equivalent to 25 mg of tissue extract was transferred to a new tube, and 250 µL of 80% methanol containing 1 µL of stable isotope-labeled internal amino acid standard mix was added. Metabolites were concentrated using a SpeedVac until dry. For plasma metabolite analysis, 10 µL of plasma was added to 250 µL of 80% methanol containing 1 µL of stable isotope-labeled internal amino acid standard mix, vortexed at 4C for 10 minutes, and centrifuged. 0.9 ml of supernatant was transferred to a new tube and concentrated using a SpeedVac until dry. Metabolites were reconstituted into 25-50 µL of water, vortexed, centrifuged, and transferred to vials for analysis by LCMS. LCMS was performed using a ZIC-pHILIC LC column coupled to a Vanquish LC and a flow gradient consisting of 10 mM ammonium carbonate in water and pure acetonitrile. 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. Data files are labeled with brain, liver, and plasma followed by a number. The number represents a mouse. Samples 1-3 are from PBS-treated mice and samples 4-6 are from LOX-treated mice. |
| 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: | PR002184 |
| Project DOI: | doi: 10.21228/M89V56 |
| Project Title: | Restricting lysine normalizes toxic catabolites associated with ALDH7A1-deficiency in cells and mice |
| Project Type: | Manuscript |
| Project Summary: | Lysine metabolism converges at α-aminoadipic semialdehyde dehydrogenase (ALDH7A1). Rare loss-of-function mutations in ALDH7A1 cause a toxic accumulation of lysine catabolites, including piperideine 6-carboxylate (P6C), that are thought to cause fatal seizures in children unless strictly managed with dietary lysine reduction. In this study, we perform metabolomics and expression analysis of tissues from Aldh7a1-deficient mice, which reveal tissue-specific differences in lysine metabolism and other metabolic pathways. We also develop a fluorescent biosensor to characterize lysine transporter activity and identify competitive substrates that reduce the accumulation of lysine catabolites in ALDH7A1-deficient HEK293 cells. Lastly, we show that intravenous administration of lysine α-oxidase from Trichoderma viride reduces lysine and P6C levels by >80% in mice. Our results improve our understanding of lysine metabolism and make inroads towards improving therapeutic strategies for lysine catabolic disorders. |
| 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: | SU003677 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Age Or Age Range: | 10-17 week |
| Gender: | Female |
| Animal Feed: | 0.9% w/w lysine, 18 ppm pyridoxine (standard diets) |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype | Treatment | Sample source |
|---|---|---|---|---|
| SA387786 | Brain_LOX_2 | Aldh7a1-/- | LOX (150 U/kg, intravenous) | Brain |
| SA387787 | Brain_LOX_3 | Aldh7a1-/- | LOX (150 U/kg, intravenous) | Brain |
| SA387788 | Brain_LOX_1 | Aldh7a1-/- | LOX (150 U/kg, intravenous) | Brain |
| SA387789 | Liver_LOX_2 | Aldh7a1-/- | LOX (150 U/kg, intravenous) | Liver |
| SA387790 | Liver_LOX_1 | Aldh7a1-/- | LOX (150 U/kg, intravenous) | Liver |
| SA387791 | Liver_LOX_3 | Aldh7a1-/- | LOX (150 U/kg, intravenous) | Liver |
| SA387792 | Plasma_LOX_2 | Aldh7a1-/- | LOX (150 U/kg, intravenous) | Plasma |
| SA387793 | Plasma_LOX_3 | Aldh7a1-/- | LOX (150 U/kg, intravenous) | Plasma |
| SA387794 | Plasma_LOX_1 | Aldh7a1-/- | LOX (150 U/kg, intravenous) | Plasma |
| SA387795 | Brain_PBS_1 | Aldh7a1-/- | PBS | Brain |
| SA387796 | Brain_PBS_2 | Aldh7a1-/- | PBS | Brain |
| SA387797 | Brain_PBS_3 | Aldh7a1-/- | PBS | Brain |
| SA387798 | Liver_PBS_1 | Aldh7a1-/- | PBS | Liver |
| SA387799 | Liver_PBS_2 | Aldh7a1-/- | PBS | Liver |
| SA387800 | Liver_PBS_3 | Aldh7a1-/- | PBS | Liver |
| SA387801 | Plasma_PBS_2 | Aldh7a1-/- | PBS | Plasma |
| SA387802 | Plasma_PBS_3 | Aldh7a1-/- | PBS | Plasma |
| SA387803 | Plasma_PBS_1 | Aldh7a1-/- | PBS | Plasma |
| Showing results 1 to 18 of 18 |
Collection:
| Collection ID: | CO003670 |
| Collection Summary: | Liver, brain, or plasma samples collected from mice treated with PBS or Lysine alpha-oxidase (150 U/kg) as a single intravenous injection. Tissues collected 7-hours after injection. |
| Sample Type: | Tissue or Plasma |
Treatment:
| Treatment ID: | TR003686 |
| Treatment Summary: | PBS or Lysine alpha-oxidase (150 U/kg) |
| Treatment Route: | intravenous |
| Treatment Vehicle: | PBS |
Sample Preparation:
| Sampleprep ID: | SP003684 |
| 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 | AN005831 | AN005832 |
|---|---|---|
| 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: | CH004431 |
| 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: | MS005551 |
| Analysis ID: | AN005831 |
| 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: | MS005552 |
| Analysis ID: | AN005832 |
| 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 |
