Summary of Study ST001744
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 PR001117. The data can be accessed directly via it's Project DOI: 10.21228/M89X2R 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 | ST001744 |
| Study Title | X13CMS: Global Tracking of Isotopic Labels in Untargeted Metabolomics |
| Study Type | Untargeted metabolomics |
| Study Summary | Studies of isotopically labeled compounds have been fundamental to understanding metabolic pathways and fluxes. They have traditionally, however, been used in conjunction with targeted analyses that identify and quantify a limited number of labeled downstream metabolites. Here we describe an alternative workflow that leverages recent advances in untargeted metabolomic technologies to track the fates of isotopically labeled metabolites in a global, unbiased manner. This untargeted approach can be applied to discover novel biochemical pathways and characterize changes in the fates of labeled metabolites as a function of altered biological conditions such as disease. To facilitate the data analysis, we introduce X13CMS, an extension of the widely used mass spectrometry-based metabolomic software package XCMS. X13CMS uses the XCMS platform to detect metabolite peaks and perform retention-time alignment in liquid chromatography/mass spectrometry (LC/MS) data. With the use of the XCMS output, the program then identifies isotopologue groups that correspond to isotopically labeled compounds. The retrieval of these groups is done without any a priori knowledge besides the following input parameters: (i) the mass difference between the unlabeled and labeled isotopes, (ii) the mass accuracy of the instrument used in the analysis, and (iii) the estimated retention-time reproducibility of the chromatographic method. Despite its name, X13CMS can be used to track any isotopic label. Additionally, it detects differential labeling patterns in biological samples collected from parallel control and experimental conditions. We validated the ability of X13CMS to accurately retrieve labeled metabolites from complex biological matrices both with targeted LC/MS/MS analysis of a subset of the hits identified by the program and with labeled standards spiked into cell extracts. We demonstrate the full functionality of X13CMS with an analysis of cultured rat astrocytes treated with uniformly labeled (U-)13C-glucose during lipopolysaccharide (LPS) challenge. Our results show that out of 223 isotopologue groups enriched from U-13C-glucose, 95 have statistically significant differential labeling patterns in astrocytes challenged with LPS compared to unchallenged control cells. Only two of these groups overlap with the 32 differentially regulated peaks identified by XCMS, indicating that X13CMS uncovers different and complementary information from untargeted metabolomic studies. Like XCMS, X13CMS is implemented in R. It is available from our laboratory website at http://pattilab.wustl.edu/x13cms.php. |
| Institute | Washington University in St. Louis |
| Last Name | Cho |
| First Name | Kevin |
| Address | 1 Brookings Drive, Campus Box 1134, St. Louis, MO, 63130, USA |
| kevin.cho@wustl.edu | |
| Phone | 314-935-8813 |
| Submit Date | 2021-03-16 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzdata.xml |
| Analysis Type Detail | LC-MS |
| Release Date | 2021-04-20 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001117 |
| Project DOI: | doi: 10.21228/M89X2R |
| Project Title: | X13CMS: Global Tracking of Isotopic Labels in Untargeted Metabolomics |
| Project Type: | Untargeted metabolomics |
| Project Summary: | Studies of isotopically labeled compounds have been fundamental to understanding metabolic pathways and fluxes. They have traditionally, however, been used in conjunction with targeted analyses that identify and quantify a limited number of labeled downstream metabolites. Here we describe an alternative workflow that leverages recent advances in untargeted metabolomic technologies to track the fates of isotopically labeled metabolites in a global, unbiased manner. This untargeted approach can be applied to discover novel biochemical pathways and characterize changes in the fates of labeled metabolites as a function of altered biological conditions such as disease. To facilitate the data analysis, we introduce X13CMS, an extension of the widely used mass spectrometry-based metabolomic software package XCMS. X13CMS uses the XCMS platform to detect metabolite peaks and perform retention-time alignment in liquid chromatography/mass spectrometry (LC/MS) data. With the use of the XCMS output, the program then identifies isotopologue groups that correspond to isotopically labeled compounds. The retrieval of these groups is done without any a priori knowledge besides the following input parameters: (i) the mass difference between the unlabeled and labeled isotopes, (ii) the mass accuracy of the instrument used in the analysis, and (iii) the estimated retention-time reproducibility of the chromatographic method. Despite its name, X13CMS can be used to track any isotopic label. Additionally, it detects differential labeling patterns in biological samples collected from parallel control and experimental conditions. We validated the ability of X13CMS to accurately retrieve labeled metabolites from complex biological matrices both with targeted LC/MS/MS analysis of a subset of the hits identified by the program and with labeled standards spiked into cell extracts. We demonstrate the full functionality of X13CMS with an analysis of cultured rat astrocytes treated with uniformly labeled (U-)13C-glucose during lipopolysaccharide (LPS) challenge. Our results show that out of 223 isotopologue groups enriched from U-13C-glucose, 95 have statistically significant differential labeling patterns in astrocytes challenged with LPS compared to unchallenged control cells. Only two of these groups overlap with the 32 differentially regulated peaks identified by XCMS, indicating that X13CMS uncovers different and complementary information from untargeted metabolomic studies. Like XCMS, X13CMS is implemented in R. It is available from our laboratory website at http://pattilab.wustl.edu/x13cms.php |
| Institute: | Washington University in St. Louis |
| Department: | Chemistry |
| Laboratory: | Patti Lab |
| Last Name: | Cho |
| First Name: | Kevin |
| Address: | 1 Brookings Drive, Campus Box 1134, St. Louis, MO, 63130, USA |
| Email: | kevin.cho@wustl.edu |
| Phone: | 314-935-8813 |
| Funding Source: | NIH |
| Publications: | https://doi.org/10.1021/ac403384n |
Subject:
| Subject ID: | SU001821 |
| Subject Type: | Cultured cells |
| Subject Species: | Rattus norvegicus |
| Taxonomy ID: | 10116 |
Factors:
Subject type: Cultured cells; Subject species: Rattus norvegicus (Factor headings shown in green)
| mb_sample_id | local_sample_id | LPS (ug/mL) | Glucose |
|---|---|---|---|
| SA163295 | LPS12C1 | 1 | U-12C Glucose |
| SA163296 | LPS12C3 | 1 | U-12C Glucose |
| SA163297 | LPS12C2 | 1 | U-12C Glucose |
| SA163298 | LPS13C3 | 1 | U-13C Glucose |
| SA163299 | LPS13C1 | 1 | U-13C Glucose |
| SA163300 | LPS13C2 | 1 | U-13C Glucose |
| SA163289 | Ctrl12C1 | - | U-12C Glucose |
| SA163290 | Ctrl12C3 | - | U-12C Glucose |
| SA163291 | Ctrl12C2 | - | U-12C Glucose |
| SA163292 | Ctrl13C2 | - | U-13C Glucose |
| SA163293 | Ctrl13C3 | - | U-13C Glucose |
| SA163294 | Ctrl13C1 | - | U-13C Glucose |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO001814 |
| Collection Summary: | Immortalized rat astrocytes (CRL-2005) |
| Sample Type: | Fibroblasts |
Treatment:
| Treatment ID: | TR001834 |
| Treatment Summary: | high-glucose DMEM (Gibco) containing 10% FBS and 1% penicillin/streptomycin (Gibco). Isotopic labeling was achieved through 30 min treatments with media containing 4.5 g/L U-13C-glucose (100% of the total glucose content; Cambridge Isotopes); parallel cultures were treated with 4.5 g/L natural-abundance glucose. Simultaneous to the introduction of the labeled substrate, subsets of both unlabeled and labeled cultures were treated with LPS (added to culture media to a final concentration of 1 μg/mL) for the duration of the labeling protocol. |
Sample Preparation:
| Sampleprep ID: | SP001827 |
| Sampleprep Summary: | Cells were then washed with phosphate buffer solution and high-performance liquid chromatography (HPLC)-grade water, quenched with 1 mL cold HPLC-grade methanol, scraped from the plate, and pelleted. Pellets were dried on a SpeedVac and subsequently lyophilized. Dried samples were weighed out and extracted with the solvent volumes adjusted to maintain a ratio of 1 mL of solvent per 1 mg of dried cellular material. The final volume of reconstitution solvent was adjusted to 100 μL per 1 mg of dried material. All cell-culture conditions (unlabeled versus labeled, control versus LPSstimulated) were sampled in triplicate. |
Combined analysis:
| Analysis ID | AN002837 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Agilent 1200 |
| Column | Luna Aminopropyl |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Agilent 6520 QTOF |
| Ion Mode | NEGATIVE |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH002100 |
| Instrument Name: | Agilent 1200 |
| Column Name: | Luna Aminopropyl |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002630 |
| Analysis ID: | AN002837 |
| Instrument Name: | Agilent 6520 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | Mass spectrometry (MS) detection was carried out on an Agilent 6520 Q-TOF in negative ESI (electrospray ionization) mode with the following settings: gas temperature 325 °C, drying gas 5 L/min, nebulizer 15 psi, fragmentor 120 V, skimmer 65 V, capillary voltage −3500 V, and scan rate 1.06 spectra/s. Tandem MS (MS/MS) analyses were carried out with identical ESI parameters, and the following fragmentation and precursor ion selection settings: collision energy 10 V, precursor isolation window 1.3 amu, and scan rate 1.00 spectra/s. X13CMS R-Package was used for data processing |
| Ion Mode: | NEGATIVE |
