#METABOLOMICS WORKBENCH kevin.cho@wustl.edu_20210316_173546 DATATRACK_ID:2528 STUDY_ID:ST001744 ANALYSIS_ID:AN002837 PROJECT_ID:PR001117 VERSION 1 CREATED_ON April 15, 2021, 5:10 pm #PROJECT PR:PROJECT_TITLE X13CMS: Global Tracking of Isotopic Labels in Untargeted Metabolomics PR:PROJECT_TYPE Untargeted metabolomics PR:PROJECT_SUMMARY Studies of isotopically labeled compounds have been fundamental to understanding PR:PROJECT_SUMMARY metabolic pathways and fluxes. They have traditionally, however, been used in PR:PROJECT_SUMMARY conjunction with targeted analyses that identify and quantify a limited number PR:PROJECT_SUMMARY of labeled downstream metabolites. Here we describe an alternative workflow that PR:PROJECT_SUMMARY leverages recent advances in untargeted metabolomic technologies to track the PR:PROJECT_SUMMARY fates of isotopically labeled metabolites in a global, unbiased manner. This PR:PROJECT_SUMMARY untargeted approach can be applied to discover novel biochemical pathways and PR:PROJECT_SUMMARY characterize changes in the fates of labeled metabolites as a function of PR:PROJECT_SUMMARY altered biological conditions such as disease. To facilitate the data analysis, PR:PROJECT_SUMMARY we introduce X13CMS, an extension of the widely used mass spectrometry-based PR:PROJECT_SUMMARY metabolomic software package XCMS. X13CMS uses the XCMS platform to detect PR:PROJECT_SUMMARY metabolite peaks and perform retention-time alignment in liquid PR:PROJECT_SUMMARY chromatography/mass spectrometry (LC/MS) data. With the use of the XCMS output, PR:PROJECT_SUMMARY the program then identifies isotopologue groups that correspond to isotopically PR:PROJECT_SUMMARY labeled compounds. The retrieval of these groups is done without any a priori PR:PROJECT_SUMMARY knowledge besides the following input parameters: (i) the mass difference PR:PROJECT_SUMMARY between the unlabeled and labeled isotopes, (ii) the mass accuracy of the PR:PROJECT_SUMMARY instrument used in the analysis, and (iii) the estimated retention-time PR:PROJECT_SUMMARY reproducibility of the chromatographic method. Despite its name, X13CMS can be PR:PROJECT_SUMMARY used to track any isotopic label. Additionally, it detects differential labeling PR:PROJECT_SUMMARY patterns in biological samples collected from parallel control and experimental PR:PROJECT_SUMMARY conditions. We validated the ability of X13CMS to accurately retrieve labeled PR:PROJECT_SUMMARY metabolites from complex biological matrices both with targeted LC/MS/MS PR:PROJECT_SUMMARY analysis of a subset of the hits identified by the program and with labeled PR:PROJECT_SUMMARY standards spiked into cell extracts. We demonstrate the full functionality of PR:PROJECT_SUMMARY X13CMS with an analysis of cultured rat astrocytes treated with uniformly PR:PROJECT_SUMMARY labeled (U-)13C-glucose during lipopolysaccharide (LPS) challenge. Our results PR:PROJECT_SUMMARY show that out of 223 isotopologue groups enriched from U-13C-glucose, 95 have PR:PROJECT_SUMMARY statistically significant differential labeling patterns in astrocytes PR:PROJECT_SUMMARY challenged with LPS compared to unchallenged control cells. Only two of these PR:PROJECT_SUMMARY groups overlap with the 32 differentially regulated peaks identified by XCMS, PR:PROJECT_SUMMARY indicating that X13CMS uncovers different and complementary information from PR:PROJECT_SUMMARY untargeted metabolomic studies. Like XCMS, X13CMS is implemented in R. It is PR:PROJECT_SUMMARY available from our laboratory website at http://pattilab.wustl.edu/x13cms.php PR:INSTITUTE Washington University in St. Louis PR:DEPARTMENT Chemistry PR:LABORATORY Patti Lab PR:LAST_NAME Cho PR:FIRST_NAME Kevin PR:ADDRESS 1 Brookings Drive, Campus Box 1134, St. Louis, MO, 63130, USA PR:EMAIL kevin.cho@wustl.edu PR:PHONE 314-935-8813 PR:FUNDING_SOURCE NIH PR:PUBLICATIONS https://doi.org/10.1021/ac403384n #STUDY ST:STUDY_TITLE X13CMS: Global Tracking of Isotopic Labels in Untargeted Metabolomics ST:STUDY_TYPE Untargeted metabolomics ST:STUDY_SUMMARY Studies of isotopically labeled compounds have been fundamental to understanding ST:STUDY_SUMMARY metabolic pathways and fluxes. They have traditionally, however, been used in ST:STUDY_SUMMARY conjunction with targeted analyses that identify and quantify a limited number ST:STUDY_SUMMARY of labeled downstream metabolites. Here we describe an alternative workflow that ST:STUDY_SUMMARY leverages recent advances in untargeted metabolomic technologies to track the ST:STUDY_SUMMARY fates of isotopically labeled metabolites in a global, unbiased manner. This ST:STUDY_SUMMARY untargeted approach can be applied to discover novel biochemical pathways and ST:STUDY_SUMMARY characterize changes in the fates of labeled metabolites as a function of ST:STUDY_SUMMARY altered biological conditions such as disease. To facilitate the data analysis, ST:STUDY_SUMMARY we introduce X13CMS, an extension of the widely used mass spectrometry-based ST:STUDY_SUMMARY metabolomic software package XCMS. X13CMS uses the XCMS platform to detect ST:STUDY_SUMMARY metabolite peaks and perform retention-time alignment in liquid ST:STUDY_SUMMARY chromatography/mass spectrometry (LC/MS) data. With the use of the XCMS output, ST:STUDY_SUMMARY the program then identifies isotopologue groups that correspond to isotopically ST:STUDY_SUMMARY labeled compounds. The retrieval of these groups is done without any a priori ST:STUDY_SUMMARY knowledge besides the following input parameters: (i) the mass difference ST:STUDY_SUMMARY between the unlabeled and labeled isotopes, (ii) the mass accuracy of the ST:STUDY_SUMMARY instrument used in the analysis, and (iii) the estimated retention-time ST:STUDY_SUMMARY reproducibility of the chromatographic method. Despite its name, X13CMS can be ST:STUDY_SUMMARY used to track any isotopic label. Additionally, it detects differential labeling ST:STUDY_SUMMARY patterns in biological samples collected from parallel control and experimental ST:STUDY_SUMMARY conditions. We validated the ability of X13CMS to accurately retrieve labeled ST:STUDY_SUMMARY metabolites from complex biological matrices both with targeted LC/MS/MS ST:STUDY_SUMMARY analysis of a subset of the hits identified by the program and with labeled ST:STUDY_SUMMARY standards spiked into cell extracts. We demonstrate the full functionality of ST:STUDY_SUMMARY X13CMS with an analysis of cultured rat astrocytes treated with uniformly ST:STUDY_SUMMARY labeled (U-)13C-glucose during lipopolysaccharide (LPS) challenge. Our results ST:STUDY_SUMMARY show that out of 223 isotopologue groups enriched from U-13C-glucose, 95 have ST:STUDY_SUMMARY statistically significant differential labeling patterns in astrocytes ST:STUDY_SUMMARY challenged with LPS compared to unchallenged control cells. Only two of these ST:STUDY_SUMMARY groups overlap with the 32 differentially regulated peaks identified by XCMS, ST:STUDY_SUMMARY indicating that X13CMS uncovers different and complementary information from ST:STUDY_SUMMARY untargeted metabolomic studies. Like XCMS, X13CMS is implemented in R. It is ST:STUDY_SUMMARY available from our laboratory website at http://pattilab.wustl.edu/x13cms.php. ST:INSTITUTE Washington University, St. Louis ST:LAST_NAME Cho ST:FIRST_NAME Kevin ST:ADDRESS 1 Brookings Drive, Campus Box 1134, St. Louis, MO, 63130, USA ST:EMAIL kevin.cho@wustl.edu ST:PHONE 314-935-8813 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10116 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS Control Ctrl12C1 LPS (ug/mL):- | Glucose:U-12C Glucose RAW_FILE_NAME=Ctrl12C1.mzdata.xml SUBJECT_SAMPLE_FACTORS Control Ctrl12C2 LPS (ug/mL):- | Glucose:U-12C Glucose RAW_FILE_NAME=Ctrl12C2.mzdata.xml SUBJECT_SAMPLE_FACTORS Control Ctrl12C3 LPS (ug/mL):- | Glucose:U-12C Glucose RAW_FILE_NAME=Ctrl12C3.mzdata.xml SUBJECT_SAMPLE_FACTORS LPS LPS12C1 LPS (ug/mL):1 | Glucose:U-12C Glucose RAW_FILE_NAME=LPS12C1.mzdata.xml SUBJECT_SAMPLE_FACTORS LPS LPS12C2 LPS (ug/mL):1 | Glucose:U-12C Glucose RAW_FILE_NAME=LPS12C2.mzdata.xml SUBJECT_SAMPLE_FACTORS LPS LPS12C3 LPS (ug/mL):1 | Glucose:U-12C Glucose RAW_FILE_NAME=LPS12C3.mzdata.xml SUBJECT_SAMPLE_FACTORS Control Ctrl13C1 LPS (ug/mL):- | Glucose:U-13C Glucose RAW_FILE_NAME=Ctrl13C1.mzdata.xml SUBJECT_SAMPLE_FACTORS Control Ctrl13C2 LPS (ug/mL):- | Glucose:U-13C Glucose RAW_FILE_NAME=Ctrl13C2.mzdata.xml SUBJECT_SAMPLE_FACTORS Control Ctrl13C3 LPS (ug/mL):- | Glucose:U-13C Glucose RAW_FILE_NAME=Ctrl13C3.mzdata.xml SUBJECT_SAMPLE_FACTORS LPS LPS13C1 LPS (ug/mL):1 | Glucose:U-13C Glucose RAW_FILE_NAME=LPS13C1.mzdata.xml SUBJECT_SAMPLE_FACTORS LPS LPS13C2 LPS (ug/mL):1 | Glucose:U-13C Glucose RAW_FILE_NAME=LPS13C2.mzdata.xml SUBJECT_SAMPLE_FACTORS LPS LPS13C3 LPS (ug/mL):1 | Glucose:U-13C Glucose RAW_FILE_NAME=LPS13C3.mzdata.xml #COLLECTION CO:COLLECTION_SUMMARY Immortalized rat astrocytes (CRL-2005) CO:SAMPLE_TYPE Fibroblasts #TREATMENT TR:TREATMENT_SUMMARY high-glucose DMEM (Gibco) containing 10% FBS and 1% penicillin/streptomycin TR:TREATMENT_SUMMARY (Gibco). Isotopic labeling was achieved through 30 min treatments with media TR:TREATMENT_SUMMARY containing 4.5 g/L U-13C-glucose (100% of the total glucose content; Cambridge TR:TREATMENT_SUMMARY Isotopes); parallel cultures were treated with 4.5 g/L natural-abundance TR:TREATMENT_SUMMARY glucose. Simultaneous to the introduction of the labeled substrate, subsets of TR:TREATMENT_SUMMARY both unlabeled and labeled cultures were treated with LPS (added to culture TR:TREATMENT_SUMMARY media to a final concentration of 1 μg/mL) for the duration of the labeling TR:TREATMENT_SUMMARY protocol. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells were then washed with phosphate buffer solution and high-performance SP:SAMPLEPREP_SUMMARY liquid chromatography (HPLC)-grade water, quenched with 1 mL cold HPLC-grade SP:SAMPLEPREP_SUMMARY methanol, scraped from the plate, and pelleted. Pellets were dried on a SpeedVac SP:SAMPLEPREP_SUMMARY and subsequently lyophilized. Dried samples were weighed out and extracted with SP:SAMPLEPREP_SUMMARY the solvent volumes adjusted to maintain a ratio of 1 mL of solvent per 1 mg of SP:SAMPLEPREP_SUMMARY dried cellular material. The final volume of reconstitution solvent was adjusted SP:SAMPLEPREP_SUMMARY to 100 μL per 1 mg of dried material. All cell-culture conditions (unlabeled SP:SAMPLEPREP_SUMMARY versus labeled, control versus LPSstimulated) were sampled in triplicate. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1200 CH:COLUMN_NAME Luna Aminopropyl #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6520 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Mass spectrometry (MS) detection was carried out on an Agilent 6520 Q-TOF in MS:MS_COMMENTS negative ESI (electrospray ionization) mode with the following settings: gas MS:MS_COMMENTS temperature 325 °C, drying gas 5 L/min, nebulizer 15 psi, fragmentor 120 V, MS:MS_COMMENTS skimmer 65 V, capillary voltage −3500 V, and scan rate 1.06 spectra/s. Tandem MS:MS_COMMENTS MS (MS/MS) analyses were carried out with identical ESI parameters, and the MS:MS_COMMENTS following fragmentation and precursor ion selection settings: collision energy MS:MS_COMMENTS 10 V, precursor isolation window 1.3 amu, and scan rate 1.00 spectra/s. X13CMS MS:MS_COMMENTS R-Package was used for data processing MS:MS_RESULTS_FILE ST001744_AN002837_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Seconds #END