Summary of Study ST001608

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 PR001033. The data can be accessed directly via it's Project DOI: 10.21228/M85M45 This work is supported by NIH grant, U2C- DK119886.

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Study IDST001608
Study TitleComparing gas chromatography with time-of-flight, quadrupole time-of-flight and quadrupole mass spectrometry for stable isotope tracing (part-I)
Study SummaryStable isotope tracers are applied in vivo and in vitro studies to reveal the activity of enzymes and intracellular metabolic pathways. Most often, such tracers are used with gas chromatography coupled to mass spectrometry (GC-MS) due to its ease of operation and reproducible mass spectral databases. Differences in isotope tracer performance of classic GC-quadrupole MS instrument and newer time-of-flight instruments are not well-studied. Here, we used three commercially available instruments for the analysis of identical samples from a stable isotope labeling study that used [U-13C6] d-glucose to investigate the metabolism of Rothia mucilaginosa with respect to 29 amino acids and hydroxyl acids involved in primary metabolism. Overall, all three GC-MS instruments (low-resolution GC-SQ-MS, low-resolution GC-TOF-MS, and high-resolution GC-Q-TOF-MS) can be used to perform stable isotope tracing studies for glycolytic intermediates, TCA metabolites and amino acids, yielding similar biological results, with high-resolution GC-Q-TOF-MS offering additional capabilities to identify chemical structures of unknown compounds that might show significant isotope enrichments in biological studies.
Institute
University of California, Davis
LaboratoryOliver Fiehn
Last NameZhang
First NameYing
AddressWest Coast Metabolomics Center, University of California, Davis, 95616, CA, USA
Emailythzhang@ucdavis.edu
Phone+1-530-754-8258
Submit Date2020-11-19
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailGC-MS
Release Date2021-05-19
Release Version1
Ying Zhang Ying Zhang
https://dx.doi.org/10.21228/M85M45
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001033
Project DOI:doi: 10.21228/M85M45
Project Title:Comparing gas chromatography with time-of-flight, quadrupole time-of-light and quadrupole mass spectrometry for stable isotope tracing
Project Summary:Stable isotope tracers are applied in vivo and in vitro studies to reveal the activity of enzymes and intracellular metabolic pathways. Most often, such tracers are used with gas chromatography coupled to mass spectrometry (GC-MS) due to its ease of operation and reproducible mass spectral databases. Differences in isotope tracer performance of classic GC-quadrupole MS instrument and newer time-of-flight instruments are not well-studied. Here, we used three commercially available instruments for the analysis of identical samples from a stable isotope labeling study that used [U-13C6] d-glucose to investigate the metabolism of Rothia mucilaginosa with respect to 29 amino acids and hydroxyl acids involved in primary metabolism. Overall, all three GC-MS instruments (low-resolution GC-SQ-MS, low-resolution GC-TOF-MS, and high-resolution GC-Q-TOF-MS) can be used to perform stable isotope tracing studies for glycolytic intermediates, TCA metabolites and amino acids, yielding similar biological results, with high-resolution GC-Q-TOF-MS offering additional capabilities to identify chemical structures of unknown compounds that might show significant isotope enrichments in biological studies.
Institute:University of California, Davis
Laboratory:Oliver Fiehn
Last Name:Ying
First Name:Zhang
Address:West Coast Metabolomics Center, University of California, Davis
Email:ythzhang@ucdavis.edu
Phone:+1-530-754-8258

Subject:

Subject ID:SU001685
Subject Type:Bacteria
Subject Species:Rothia mucilaginosa
Taxonomy ID:43675
Genotype Strain:RmFLR01

Factors:

Subject type: Bacteria; Subject species: Rothia mucilaginosa (Factor headings shown in green)

mb_sample_id local_sample_id Condition
SA136482Rothia41_10Ambient | Collection time:12h
SA136483Rothia42_01Ambient | Collection time:12h
SA136484Rothia41_09Ambient | Collection time:12h
SA136485Rothia41_08Ambient | Collection time:12h
SA136486Rothia41_06Ambient | Collection time:12h
SA136487Rothia41_07Ambient | Collection time:12h
SA136488Rothia42_02Ambient | Collection time:12h
SA136489Rothia42_04Ambient | Collection time:12h
SA136490Rothia42_08Ambient | Collection time:12h
SA136491Rothia42_09Ambient | Collection time:12h
SA136492Rothia42_07Ambient | Collection time:12h
SA136493Rothia42_06Ambient | Collection time:12h
SA136494Rothia41_05Ambient | Collection time:12h
SA136495Rothia42_05Ambient | Collection time:12h
SA136496Rothia42_03Ambient | Collection time:12h
SA136497Rothia40_05Ambient | Collection time:12h
SA136498Rothia40_04Ambient | Collection time:12h
SA136499Rothia40_06Ambient | Collection time:12h
SA136500Rothia40_03Ambient | Collection time:12h
SA136501Rothia40_02Ambient | Collection time:12h
SA136502Rothia42_10Ambient | Collection time:12h
SA136503Rothia40_01Ambient | Collection time:12h
SA136504Rothia40_07Ambient | Collection time:12h
SA136505Rothia40_08Ambient | Collection time:12h
SA136506Rothia41_02Ambient | Collection time:12h
SA136507Rothia41_03Ambient | Collection time:12h
SA136508Rothia41_01Ambient | Collection time:12h
SA136509Rothia40_10Ambient | Collection time:12h
SA136510Rothia40_09Ambient | Collection time:12h
SA136511Rothia41_04Ambient | Collection time:12h
SA136512Rothia43_07Ambient | Collection time:24h
SA136513Rothia45_01Ambient | Collection time:24h
SA136514Rothia45_02Ambient | Collection time:24h
SA136515Rothia44_10Ambient | Collection time:24h
SA136516Rothia44_09Ambient | Collection time:24h
SA136517Rothia44_07Ambient | Collection time:24h
SA136518Rothia44_08Ambient | Collection time:24h
SA136519Rothia45_03Ambient | Collection time:24h
SA136520Rothia45_04Ambient | Collection time:24h
SA136521Rothia45_09Ambient | Collection time:24h
SA136522Rothia45_10Ambient | Collection time:24h
SA136523Rothia43_01Ambient | Collection time:24h
SA136524Rothia45_07Ambient | Collection time:24h
SA136525Rothia45_05Ambient | Collection time:24h
SA136526Rothia45_06Ambient | Collection time:24h
SA136527Rothia44_06Ambient | Collection time:24h
SA136528Rothia45_08Ambient | Collection time:24h
SA136529Rothia43_05Ambient | Collection time:24h
SA136530Rothia43_06Ambient | Collection time:24h
SA136531Rothia43_04Ambient | Collection time:24h
SA136532Rothia43_02Ambient | Collection time:24h
SA136533Rothia44_05Ambient | Collection time:24h
SA136534Rothia43_08Ambient | Collection time:24h
SA136535Rothia43_03Ambient | Collection time:24h
SA136536Rothia44_04Ambient | Collection time:24h
SA136537Rothia44_03Ambient | Collection time:24h
SA136538Rothia44_02Ambient | Collection time:24h
SA136539Rothia44_01Ambient | Collection time:24h
SA136540Rothia43_09Ambient | Collection time:24h
SA136541Rothia43_10Ambient | Collection time:24h
SA136542Rothia35_02Ambient | Collection time:4h
SA136543Rothia35_01Ambient | Collection time:4h
SA136544Rothia35_05Ambient | Collection time:4h
SA136545Rothia35_06Ambient | Collection time:4h
SA136546Rothia35_04Ambient | Collection time:4h
SA136547Rothia35_03Ambient | Collection time:4h
SA136548Rothia34_08Ambient | Collection time:4h
SA136549Rothia35_07Ambient | Collection time:4h
SA136550Rothia34_05Ambient | Collection time:4h
SA136551Rothia34_06Ambient | Collection time:4h
SA136552Rothia34_07Ambient | Collection time:4h
SA136553Rothia34_09Ambient | Collection time:4h
SA136554Rothia34_10Ambient | Collection time:4h
SA136555Rothia36_05Ambient | Collection time:4h
SA136556Rothia36_08Ambient | Collection time:4h
SA136557Rothia36_07Ambient | Collection time:4h
SA136558Rothia36_09Ambient | Collection time:4h
SA136559Rothia36_10Ambient | Collection time:4h
SA136560Rothia34_04Ambient | Collection time:4h
SA136561Rothia36_06Ambient | Collection time:4h
SA136562Rothia36_04Ambient | Collection time:4h
SA136563Rothia35_10Ambient | Collection time:4h
SA136564Rothia36_01Ambient | Collection time:4h
SA136565Rothia36_02Ambient | Collection time:4h
SA136566Rothia36_03Ambient | Collection time:4h
SA136567Rothia35_09Ambient | Collection time:4h
SA136568Rothia35_08Ambient | Collection time:4h
SA136569Rothia34_03Ambient | Collection time:4h
SA136570Rothia34_01Ambient | Collection time:4h
SA136571Rothia34_02Ambient | Collection time:4h
SA136572Rothia38_03Ambient | Collection time:8h
SA136573Rothia38_04Ambient | Collection time:8h
SA136574Rothia38_06Ambient | Collection time:8h
SA136575Rothia38_02Ambient | Collection time:8h
SA136576Rothia38_05Ambient | Collection time:8h
SA136577Rothia37_10Ambient | Collection time:8h
SA136578Rothia37_07Ambient | Collection time:8h
SA136579Rothia37_06Ambient | Collection time:8h
SA136580Rothia37_08Ambient | Collection time:8h
SA136581Rothia37_09Ambient | Collection time:8h
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Collection:

Collection ID:CO001678
Collection Summary:A quality control mixture of 29 unlabeled metabolite standards (Supporting information Table SI 1) was prepared to reach a final concentration of 1 mg/mL as stock solution. 50 µL aliquot from each stock solution was combined into a new tube and dried down. Then, the mixture was diluted to reach a final concentration of 50 µg/mL working solution. Rothia mucilaginosa strain RmFLR01 was isolated from a cystic fibrosis (CF) patient at the UC San Diego Adult CF Clinic. R. mucilaginosa cultures were grown in triplicates in artificial-sputum medium spiked with 100 mM [U-13C6] d-glucose (Cambridge Isotope Laboratory, Tewksbury, MA, USA) under anaerobic and aerobic conditions (5% CO2) at 37°C and harvested at 4, 8, 12 and 24 h for isotope tracer analyses.
Sample Type:Bacterial cells

Treatment:

Treatment ID:TR001698
Treatment Summary:For the 29 unlabeled standards mixture, 10 µL of the final solution was dried down for GC-MS measurement. Derivatization and data acquisition of mixture aliquots by GC-MS were reproduced on three days for inter-day precision. Dried mixtures were derivatized by adding 10 µL of 40 mg/mL methoxyamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in pyridine (Sigma-Aldrich, St. Louis, MO, USA) and shaking at 30°C for 1.5 h. Subsequently, 90 µL MTBSTFA (Sigma-Aldrich, St. Louis, MO, USA) was added with 13 fatty acid methyl esters (FAMEs) as retention index markers and shaken at 80°C for 30 min. Samples were immediately transferred to crimp top vials and injected onto each GC-MS instrument. Same samples of R. mucilaginosa cultures were extracted using published methods [19]. Samples were added 1 mL pre-chilled, degassed acetonitrile: isopropanol: water (v/v/v 3:3:2, Fisher Scientific) followed by vortexing 30 s and shaking at 4°C for 5 min. Samples were centrifuged for 2 min at 12,210 × g to precipitate debris from extracts. Supernatants were collected and split into two equal portions. One aliquot was dried to completeness in a Labconco cold trap centrifuge evaporator and then resuspended in 0.5 mL degassed acetonitrile: water (v/v 1:1, Fisher Scientific) to remove triacylglycerides. Resuspension solutions were vortexed for 30s and centrifuged for 2 min. Supernatants were transferred into clean Eppendorf tubes and dried down completely. Dried extracts were derivatized as given above.

Sample Preparation:

Sampleprep ID:SP001691
Sampleprep Summary:For the 29 unlabeled standards mixture, 10 µL of the final solution was dried down for GC-MS measurement. Derivatization and data acquisition of mixture aliquots by GC-MS were reproduced on three days for inter-day precision. Dried mixtures were derivatized by adding 10 µL of 40 mg/mL methoxyamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in pyridine (Sigma-Aldrich, St. Louis, MO, USA) and shaking at 30°C for 1.5 h. Subsequently, 90 µL MTBSTFA (Sigma-Aldrich, St. Louis, MO, USA) was added with 13 fatty acid methyl esters (FAMEs) as retention index markers and shaken at 80°C for 30 min. Samples were immediately transferred to crimp top vials and injected onto each GC-MS instrument. Same samples of R. mucilaginosa cultures were extracted using published methods [19]. Samples were added 1 mL pre-chilled, degassed acetonitrile: isopropanol: water (v/v/v 3:3:2, Fisher Scientific) followed by vortexing 30 s and shaking at 4°C for 5 min. Samples were centrifuged for 2 min at 12,210 × g to precipitate debris from extracts. Supernatants were collected and split into two equal portions. One aliquot was dried to completeness in a Labconco cold trap centrifuge evaporator and then resuspended in 0.5 mL degassed acetonitrile: water (v/v 1:1, Fisher Scientific) to remove triacylglycerides. Resuspension solutions were vortexed for 30s and centrifuged for 2 min. Supernatants were transferred into clean Eppendorf tubes and dried down completely. Dried extracts were derivatized as given above.

Combined analysis:

Analysis ID AN002641 AN002642
Analysis type MS MS
Chromatography type GC GC
Chromatography system Agilent 7890A Agilent 7890A
Column Restek Rtx-5Sil (30m x 0.25mm,0.25um) Restek Rtx-5Sil (30m x 0.25mm,0.25um)
MS Type EI EI
MS instrument type QTOF Single quadrupole
MS instrument name Agilent 7200 QTOF Agilent 5977
Ion Mode POSITIVE POSITIVE
Units peak height peak height

Chromatography:

Chromatography ID:CH001951
Chromatography Summary:an Agilent 7890 GC system installed with a Restek RTX-5Sil MS column (30m length, 0.25 mm i.d, 0.25 μM df, 95% dimethyl/5%diphenyl polysiloxane film) with an additional 10m guard column. For Q-TOF-MS analyses,GC parameters were used by injecting 1 µL of derivatized sample into the GC in splitless mode at an injection temperature of 250°C and a constant flow of 1 mL/min. The initial oven temperature was held at 60°C for 0.5 min, and ramped at a rate of 10°C/min to 325°C that was maintained for 10 min for a total run time of 37 min.
Instrument Name:Agilent 7890A
Column Name:Restek Rtx-5Sil (30m x 0.25mm,0.25um)
Chromatography Type:GC

MS:

MS ID:MS002453
Analysis ID:AN002641
Instrument Name:Agilent 7200 QTOF
Instrument Type:QTOF
MS Type:EI
MS Comments:MassHunter Quantitative Analysis B.07.00 version was used to process the data
Ion Mode:POSITIVE
  
MS ID:MS002454
Analysis ID:AN002642
Instrument Name:Agilent 5977
Instrument Type:Single quadrupole
MS Type:EI
MS Comments:MassHunter Quantitative Analysis B.07.00 version was used to process the data
Ion Mode:POSITIVE
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