Summary of Study ST001860
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 PR001173. The data can be accessed directly via it's Project DOI: 10.21228/M8341K 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 | ST001860 |
| Study Title | Spontaneous hydrolysis and spurious metabolic properties of α-ketoglutarate esters |
| Study Type | Manuscript |
| Study Summary | 8988T cells treated with methyl acetate or 1 mM of alpha-ketoglutarate disodium salt or 1 mM of dimethyl-alpha-ketoglutarate for 3 hours prior to rapid quenching of metabolism and extraction of metabolites in 80% methanol (-80°C) containing internal QC standards. |
| Institute | University of British Columbia |
| Last Name | Parker |
| First Name | Seth |
| Address | 950 W 28th Ave, 2099, Vancouver, British Columbia, Canada V6H 0B3 |
| seth.parker@bcchr.ca | |
| Phone | 6048753121 |
| Submit Date | 2021-05-26 |
| Num Groups | 3 |
| Total Subjects | 9 |
| Num Males | n/a |
| Num Females | n/a |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2021-08-04 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001173 |
| Project DOI: | doi: 10.21228/M8341K |
| Project Title: | Spontaneous hydrolysis and spurious metabolic properties of α-ketoglutarate esters |
| Project Type: | Manuscript |
| Project Summary: | α-ketoglutarate (KG), also referred to as 2-oxoglutarate, is a key intermediate of cellular metabolism with pleiotropic functions. Cell-permeable esterified analogs are widely used to study the role of KG in governing bioenergetic and amino acid metabolism and DNA, RNA, and protein hydroxylation reactions, as cellular membranes are thought to be impermeable to KG. Here we show that esterified KG analogs rapidly hydrolyze in aqueous media, yielding KG that, in contrast to prevailing assumptions, can be imported by many cell lines. Esterified KG analogs exhibit spurious KG-independent effects on cellular metabolism, including extracellular acidification, arising from rapid hydrolysis and de-protonation of α-ketoesters, and significant analog-specific inhibitory effects on glycolysis or mitochondrial respiration. In many cell lines, imported KG metabolizes to succinate in the cytosol, and we observe minimal KG utilization for mitochondrial metabolism in normal culture conditions. These findings demonstrate that nuclear and cytosolic KG-dependent reactions may derive KG from functionally distinct subcellular pools and sources. |
| Institute: | University of British Columbia |
| Department: | Biochemistry & Molecular Biology |
| Last Name: | Parker |
| First Name: | Seth |
| Address: | 950 W 28th Ave, Room 2099, Vancouver, British Columbia, Canada V6H 0B3 |
| Email: | seth.parker@bcchr.ca |
| Phone: | 6048753121 |
Subject:
| Subject ID: | SU001937 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Biosource Or Supplier: | DSMZ |
| Cell Strain Details: | 8988T |
| Subject Comments: | pancreatic ductal adenocarcinoma |
| Cell Counts: | 1-2x10^6 |
| Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment |
|---|---|---|
| SA174336 | DMKG_1 | DMKG (1 mM) |
| SA174337 | DMKG_2 | DMKG (1 mM) |
| SA174338 | DMKG_3 | DMKG (1 mM) |
| SA174339 | KG_3 | KG (1 mM) |
| SA174340 | KG_2 | KG (1 mM) |
| SA174341 | KG_1 | KG (1 mM) |
| SA174342 | veh_2 | vehicle |
| SA174343 | veh_3 | vehicle |
| SA174344 | veh_1 | vehicle |
| Showing results 1 to 9 of 9 |
Collection:
| Collection ID: | CO001930 |
| Collection Summary: | Metabolites were initially extracted from samples by quickly aspirating the cell culture media and adding 1 mL of extraction buffer, consisting of 80% methanol (Fisher Scientific) and 500 nM metabolomics amino acid mix standard (Cambridge Isotope Laboratories). To effectively scale all harvested samples to equivalent volumes of extraction buffer, samples were fully dried down by Speedvac (Thermo Fisher, Waltham, MA) and reconstituted volumetrically by mixing the entire dried cell pellet sample with 1 mL of 80% methanol without QC standards in 2.0 mL screw cap vials containing ~100 µL of disruption beads (Research Products International, Mount Prospect, IL). Samples were scaled to a ratio of 1e6 cells to 1 mL of extraction solvent with all steps being carried out in a cold room. Each was homogenized for 10 cycles on a bead blaster homogenizer (Benchmark Scientific, Edison, NJ). Cycling consisted of a 30 sec homogenization time at 6 m/s followed by a 30 sec pause. Samples were subsequently spun at 21,000 x g for 3 min at 4°C. A set volume of each (450 µL) was transferred to a 1.5 mL tube and dried down by Speedvac concentration. Samples were reconstituted in 50 µL of Optima LC/MS grade water (Fisher Scientific, Waltham, MA). Samples were sonicated for 2 mins, then centrifuged at 21,000 x g for 3 min at 4°C. Twenty microliters were transferred to LC vials containing glass inserts for analysis. The remaining sample was placed in -80°C for long term storage. |
| Sample Type: | Cultured cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001949 |
| Treatment Summary: | 8988T cells were treated with methyl acetate, which is used as a vehicle, 1 mM of alpha-ketoglutarate disodium salt, or 1 mM of dimethyl-alpha-ketoglutarate (prepared in methyl acetate) for 3 hours in DMEM supplemented with 10% dialyzed fetal bovine serum and 5% penstrep |
Sample Preparation:
| Sampleprep ID: | SP001943 |
| Sampleprep Summary: | Dried samples were reconstituted in 50 µL of Optima LC/MS grade water (Fisher Scientific, Waltham, MA). Samples were sonicated for 2 mins, then centrifuged at 21,000 x g for 3 min at 4°C. Twenty microliters were transferred to LC vials containing glass inserts for analysis. The remaining sample was placed in -80°C for long term storage. |
Combined analysis:
| Analysis ID | AN003015 | AN003016 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
| Column | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | ion counts | ion counts |
Chromatography:
| Chromatography ID: | CH002234 |
| Chromatography Summary: | Samples were subjected to an LC-MS analysis to detect and quantify known peaks. A metabolite extraction was carried out on each sample by quickly aspirating experimental media and adding 1 mL of 80% methanol containing internal QC standards. The LC column was a MilliporeTM ZIC-pHILIC (2.1 x150 mm, 5 μm) coupled to a Dionex Ultimate 3000TM system and the column oven temperature was set to 25oC for the gradient elution. A flow rate of 100 μL/min was used with the following buffers; A) 10 mM ammonium carbonate in water, pH 9.0, and B) neat acetonitrile. The gradient profile was as follows; 80-20%B (0-30 min), 20-80%B (30-31 min), 80-80%B (31-42 min). Injection volume was set to 2 μL for all analyses (42 min total run time per injection). |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 25 |
| Flow Rate: | 100 uL/min |
| Solvent A: | 100% water; 10 mM ammonium carbonate, pH 9.0 |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002804 |
| Analysis ID: | AN003015 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS analyses were carried out by coupling the LC system to a Thermo Q Exactive HFTM mass spectrometer operating in heated electrospray ionization mode (HESI). Method duration was 30 min with a polarity switching data-dependent Top 5 method for both positive and negative modes. Spray voltage for both positive and negative modes was 3.5 kV and capillary temperature was set to 320oC with a sheath gas rate of 35, aux gas of 10, and max spray current of 100 μA. The full MS scan for both polarities utilized 120,000 resolution with an AGC target of 3e6 and a maximum IT of 100 ms, and the scan range was from 67-1000 m/z. Tandem MS spectra for both positive and negative mode used a resolution of 15,000, AGC target of 1e5, maximum IT of 50 ms, isolation window of 0.4 m/z, isolation offset of 0.1 m/z, fixed first mass of 50 m/z, and 3-way multiplexed normalized collision energies (nCE) of 10, 30, 80. The minimum AGC target was 1e4 with an intensity threshold of 2e5. All data were acquired in profile mode. The resulting ThermoTM RAW files were read with ThermoFisher CommonCore RawFileReader, and an in-house python script (Skeleton) was used for peak detection and quantification of all internal standards and sample peaks based on a previously established library of metabolite retention times and accurate masses adapted from the Whitehead Institute, and verified with authentic standards and/or high resolution MS/MS spectral manually curated against the NIST14MS/MS and METLIN (2017) tandem mass spectral libraries. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS002805 |
| Analysis ID: | AN003016 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | MS analyses were carried out by coupling the LC system to a Thermo Q Exactive HFTM mass spectrometer operating in heated electrospray ionization mode (HESI). Method duration was 30 min with a polarity switching data-dependent Top 5 method for both positive and negative modes. Spray voltage for both positive and negative modes was 3.5 kV and capillary temperature was set to 320oC with a sheath gas rate of 35, aux gas of 10, and max spray current of 100 μA. The full MS scan for both polarities utilized 120,000 resolution with an AGC target of 3e6 and a maximum IT of 100 ms, and the scan range was from 67-1000 m/z. Tandem MS spectra for both positive and negative mode used a resolution of 15,000, AGC target of 1e5, maximum IT of 50 ms, isolation window of 0.4 m/z, isolation offset of 0.1 m/z, fixed first mass of 50 m/z, and 3-way multiplexed normalized collision energies (nCE) of 10, 30, 80. The minimum AGC target was 1e4 with an intensity threshold of 2e5. All data were acquired in profile mode. The resulting ThermoTM RAW files were read with ThermoFisher CommonCore RawFileReader, and an in-house python script (Skeleton) was used for peak detection and quantification of all internal standards and sample peaks based on a previously established library of metabolite retention times and accurate masses adapted from the Whitehead Institute, and verified with authentic standards and/or high resolution MS/MS spectral manually curated against the NIST14MS/MS and METLIN (2017) tandem mass spectral libraries. |
| Ion Mode: | POSITIVE |
