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MTBLS213: geoRge: A Computational Tool To Detect the Presence of Stable Isotope Labeling in LC/MS-Based Untargeted Metabolomics.

Abstract

Studying the flow of chemical moieties through the complex set of metabolic reactions that happen in the cell is essential to understanding the alterations in homeostasis that occur in disease. Recently, LC/MS-based untargeted metabolomics and isotopically labeled metabolites have been used to facilitate the unbiased mapping of labeled moieties through metabolic pathways. However, due to the complexity of the resulting experimental data sets few computational tools are available for data analysis. Here we introduce geoRge, a novel computational approach capable of analyzing untargeted LC/MS data from stable isotope-labeling experiments. geoRge is written in the open language R and runs on the output structure of the XCMS package, which is in widespread use. As opposed to the few existing tools, which use labeled samples to track stable isotopes by iterating over all MS signals using the theoretical mass difference between the light and heavy isotopes, geoRge uses unlabeled and labeled biologically equivalent samples to compare isotopic distributions in the mass spectra. Isotopically enriched compounds change their isotopic distribution as compared to unlabeled compounds. This is directly reflected in a number of new m/z peaks and higher intensity peaks in the mass spectra of labeled samples relative to the unlabeled equivalents. The automated untargeted isotope annotation and relative quantification capabilities of geoRge are demonstrated by the analysis of LC/MS data from a human retinal pigment epithelium cell line (ARPE-19) grown on normal and high glucose concentrations mimicking diabetic retinopathy conditions in vitro. In addition, we compared the results of geoRge with the outcome of X(13)CMS, since both approaches rely entirely on XCMS parameters for feature selection, namely m/z and retention time values. geoRge is available as an R script at https://github.com/jcapelladesto/geoRge.

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 Authors: Jordi Capellades, Oscar Yanes

  Release date: 15-Sep-2015

 Status: Public

Organism(s)

Homo sapiens

  Study Design

untargeted metabolites

carbon tracking

CHMO:ultra-performance liquid chromatography-mass spectrometry

NCIT:Diabetic Retinopathy

  Experimental Factors

D-glucose

Label

Protocol Description
Sample collection Cells were cultured under standard conditions in DMEM/F12 (1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12), 10% fetal calf serum (FCS) and penicillin/streptomycin. ARPE19 cells from passage 2023 were used and the media was changed every 3 days. Cells grown in these conditions constitute a monolayer that retains the functionality, polarity and tight junction expression of the human RPE6. For our study, cells were seeded in Petri dishes (10 cm) at 0.4 x 10^4 cells/ml and maintained in culture for 21 days with 5.5 mM or 25 mM of D-Glucose at 37 °C under 5% (v/v) CO2 in an incubator. During the last 24 hours cells were subjected to serum deprivation (1% FCS). Serum deprived media were prepared with 5.5 mM or 25 mM of either D-Glucose or D-[U 13C] Glucose. Each condition was run in triplicate.
Extraction The culture medium was removed from cells and the dishes were placed on top of dry ice. Cells were scraped immediately and metabolites extracted into the extraction solvent by adding 2 ml of a cold mixture of chloroform/methanol (2:1 v/v). The resulting suspension was bath sonicated for 3 minutes, and 2 ml of cold water was added. Then, 1 ml of chloroform/methanol (2:1 v/v) was added to the samples and bath sonicated for 3 minutes. Cell lysates were centrifuged (5000 × g, 15 min at 4 °C) and the aqueous phase was carefully transferred into a new tube. The sample was frozen, lyophilized and stored at -80 °C until further analysis.
Chromatography LC/MS analyses were performed using an UHPLC system (1290 series, Agilent Technologies) coupled to a 6550 ESIQTOF MS (Agilent Technologies) operated in negative (ESI) electrospray ionization mode. Vials containing extracted metabolites were kept at -20 °C prior to LC/MS analysis. Metabolites were separated using an Acquity UPLC (BEH) C18 RP column (2.1 x 150 mm, 1.7 µm) and the solvent system was A2 = 1 mM ammonium fluoride in water and B2 = acetonitrile, as previously reported. The linear gradient elution started at 100% A (time 0–2 min) and finished at 100% B (10-15 min). The injection volume was 5 µl.
Mass spectrometry As mentioned in the previous section, mass spectra were collected in negative ionization mode. The instrument conditions were: gas temperature, 150 °C; drying gas, 13 l/min; nebulizer, 35 psig; fragmentor, 400 V; and skimmer, 65 V. The instrument was set to acquire over the m/z range 100–1500 in fullscan mode with an acquisition rate of 4 spectra/sec. MS/MS was performed in targeted mode, and the instrument was set to acquire over the m/z range 50–1000, with a default isolation width (the width halfmaximum of the quadrupole mass bandpass used during MS/MS precursor isolation) of 4 m/z. The collision energy was fixed at 20 V.
Data transformation LC/MS raw data files were transformed into .mzXML format using Proteowizard software and then was processed using the XCMS R package 9 10,11 to detect and align features. The parameters used in the XCMS workflow were: xcmsSet(method="centWave", ppm=30, peakwidth=c(5,20)); retcor(method="obiwarp", profStep=0.1) and group(mzwid=0.0065, minfrac=0.5, bw= 4). A feature is defined as a molecular entity with a unique m/z and a specific retention time (mzRT). XCMS analysis of these data provided different matrix containing the retention time, m/z value, and integrated peak area of each feature for every ARPE19 sample.
Metabolite identification All metabolite identifications are confirmed with level 1 of metabolomics standards initiative.
Source Name Organism Organism part Protocol REF Sample Name D-glucose Unit Label
CELL_Glc12_05mM_Normo_04 Homo sapiens ARPE-19 cell Sample collection CELL_Glc12_05mM_Normo_04 5 millimolar 12C
CELL_Glc12_05mM_Normo_05 Homo sapiens ARPE-19 cell Sample collection CELL_Glc12_05mM_Normo_05 5 millimolar 12C
CELL_Glc12_05mM_Normo_06 Homo sapiens ARPE-19 cell Sample collection CELL_Glc12_05mM_Normo_06 5 millimolar 12C
CELL_Glc12_25mM_Normo_16 Homo sapiens ARPE-19 cell Sample collection CELL_Glc12_25mM_Normo_16 25 millimolar 12C
CELL_Glc12_25mM_Normo_17 Homo sapiens ARPE-19 cell Sample collection CELL_Glc12_25mM_Normo_17 25 millimolar 12C
CELL_Glc12_25mM_Normo_18 Homo sapiens ARPE-19 cell Sample collection CELL_Glc12_25mM_Normo_18 25 millimolar 12C
CELL_Glc13_05mM_Normo_01 Homo sapiens ARPE-19 cell Sample collection CELL_Glc13_05mM_Normo_01 5 millimolar 13C
CELL_Glc13_05mM_Normo_02 Homo sapiens ARPE-19 cell Sample collection CELL_Glc13_05mM_Normo_02 5 millimolar 13C
CELL_Glc13_05mM_Normo_03 Homo sapiens ARPE-19 cell Sample collection CELL_Glc13_05mM_Normo_03 5 millimolar 13C
CELL_Glc13_25mM_Normo_13 Homo sapiens ARPE-19 cell Sample collection CELL_Glc13_25mM_Normo_13 25 millimolar 13C
CELL_Glc13_25mM_Normo_14 Homo sapiens ARPE-19 cell Sample collection CELL_Glc13_25mM_Normo_14 25 millimolar 13C
CELL_Glc13_25mM_Normo_15 Homo sapiens ARPE-19 cell Sample collection CELL_Glc13_25mM_Normo_15 25 millimolar 13C

Assay 

Assay file name: a_arpe_george_neg_metabolite_profiling_mass_spectrometry.txt
Measurement: metabolite profiling
Technology: mass spectrometry
Platform: 6540 Q-TOF LC/MS (Agilent)

Instrumentation

Sample Name Protocol REF Post Extraction Derivatization Extract Name Protocol REF Chromatography Instrument Column model Column type Labeled Extract Name Label Protocol REF Scan polarity Scan m/z range Instrument Ion source Mass analyzer MS Assay Name Raw Spectral Data File Protocol REF Normalization Name Derived Spectral Data File Protocol REF Data Transformation Name Metabolite Assignment File
CELL_Glc12_05mM_Normo_04 Extraction none Chromatography Agilent 1290 Infinity UHPLC ACQUITY UPLC BEH C18 (1.7 µm, 2.1 mm × 150 mm; Waters) reverse phase C12 Mass spectrometry negative 100-1500 Agilent 6550 iFunnel Q-TOF electrospray ionization quadrupole time-of-flight CELL_Glc12_05mM_Normo_04 CELL_Glc12_05mM_Normo_04.mzXML Data transformation Metabolite identification m_arpe_george_neg_metabolite_profiling_mass_spectrometry_v2_maf.tsv
CELL_Glc12_05mM_Normo_05 Extraction none Chromatography Agilent 1290 Infinity UHPLC ACQUITY UPLC BEH C18 (1.7 µm, 2.1 mm × 150 mm; Waters) reverse phase C12 Mass spectrometry negative 100-1500 Agilent 6550 iFunnel Q-TOF electrospray ionization quadrupole time-of-flight CELL_Glc12_05mM_Normo_05 CELL_Glc12_05mM_Normo_05.mzXML Data transformation Metabolite identification m_arpe_george_neg_metabolite_profiling_mass_spectrometry_v2_maf.tsv
CELL_Glc12_05mM_Normo_06 Extraction none Chromatography Agilent 1290 Infinity UHPLC ACQUITY UPLC BEH C18 (1.7 µm, 2.1 mm × 150 mm; Waters) reverse phase C12 Mass spectrometry negative 100-1500 Agilent 6550 iFunnel Q-TOF electrospray ionization quadrupole time-of-flight CELL_Glc12_05mM_Normo_06 CELL_Glc12_05mM_Normo_06.mzXML Data transformation Metabolite identification m_arpe_george_neg_metabolite_profiling_mass_spectrometry_v2_maf.tsv
CELL_Glc12_25mM_Normo_16 Extraction none Chromatography Agilent 1290 Infinity UHPLC ACQUITY UPLC BEH C18 (1.7 µm, 2.1 mm × 150 mm; Waters) reverse phase C12 Mass spectrometry negative 100-1500 Agilent 6550 iFunnel Q-TOF electrospray ionization quadrupole time-of-flight CELL_Glc12_25mM_Normo_16 CELL_Glc12_25mM_Normo_16.mzXML Data transformation Metabolite identification m_arpe_george_neg_metabolite_profiling_mass_spectrometry_v2_maf.tsv
CELL_Glc12_25mM_Normo_17 Extraction none Chromatography Agilent 1290 Infinity UHPLC ACQUITY UPLC BEH C18 (1.7 µm, 2.1 mm × 150 mm; Waters) reverse phase C12 Mass spectrometry negative 100-1500 Agilent 6550 iFunnel Q-TOF electrospray ionization quadrupole time-of-flight CELL_Glc12_25mM_Normo_17 CELL_Glc12_25mM_Normo_17.mzXML Data transformation Metabolite identification m_arpe_george_neg_metabolite_profiling_mass_spectrometry_v2_maf.tsv
CELL_Glc12_25mM_Normo_18 Extraction none Chromatography Agilent 1290 Infinity UHPLC ACQUITY UPLC BEH C18 (1.7 µm, 2.1 mm × 150 mm; Waters) reverse phase C12 Mass spectrometry negative 100-1500 Agilent 6550 iFunnel Q-TOF electrospray ionization quadrupole time-of-flight CELL_Glc12_25mM_Normo_18 CELL_Glc12_25mM_Normo_18.mzXML Data transformation Metabolite identification m_arpe_george_neg_metabolite_profiling_mass_spectrometry_v2_maf.tsv
CELL_Glc13_05mM_Normo_01 Extraction none Chromatography Agilent 1290 Infinity UHPLC ACQUITY UPLC BEH C18 (1.7 µm, 2.1 mm × 150 mm; Waters) reverse phase C13 Mass spectrometry negative 100-1500 Agilent 6550 iFunnel Q-TOF electrospray ionization quadrupole time-of-flight CELL_Glc13_05mM_Normo_01 CELL_Glc13_05mM_Normo_01.mzXML Data transformation Metabolite identification m_arpe_george_neg_metabolite_profiling_mass_spectrometry_v2_maf.tsv
CELL_Glc13_05mM_Normo_02 Extraction none Chromatography Agilent 1290 Infinity UHPLC ACQUITY UPLC BEH C18 (1.7 µm, 2.1 mm × 150 mm; Waters) reverse phase C13 Mass spectrometry negative 100-1500 Agilent 6550 iFunnel Q-TOF electrospray ionization quadrupole time-of-flight CELL_Glc13_05mM_Normo_02 CELL_Glc13_05mM_Normo_02.mzXML Data transformation Metabolite identification m_arpe_george_neg_metabolite_profiling_mass_spectrometry_v2_maf.tsv
CELL_Glc13_05mM_Normo_03 Extraction none Chromatography Agilent 1290 Infinity UHPLC ACQUITY UPLC BEH C18 (1.7 µm, 2.1 mm × 150 mm; Waters) reverse phase C13 Mass spectrometry negative 100-1500 Agilent 6550 iFunnel Q-TOF electrospray ionization quadrupole time-of-flight CELL_Glc13_05mM_Normo_03 CELL_Glc13_05mM_Normo_03.mzXML Data transformation Metabolite identification m_arpe_george_neg_metabolite_profiling_mass_spectrometry_v2_maf.tsv
CELL_Glc13_25mM_Normo_13 Extraction none Chromatography Agilent 1290 Infinity UHPLC ACQUITY UPLC BEH C18 (1.7 µm, 2.1 mm × 150 mm; Waters) reverse phase C13 Mass spectrometry negative 100-1500 Agilent 6550 iFunnel Q-TOF electrospray ionization quadrupole time-of-flight CELL_Glc13_25mM_Normo_13 CELL_Glc13_25mM_Normo_13.mzXML Data transformation Metabolite identification m_arpe_george_neg_metabolite_profiling_mass_spectrometry_v2_maf.tsv
CELL_Glc13_25mM_Normo_14 Extraction none Chromatography Agilent 1290 Infinity UHPLC ACQUITY UPLC BEH C18 (1.7 µm, 2.1 mm × 150 mm; Waters) reverse phase C13 Mass spectrometry negative 100-1500 Agilent 6550 iFunnel Q-TOF electrospray ionization quadrupole time-of-flight CELL_Glc13_25mM_Normo_14 CELL_Glc13_25mM_Normo_14.mzXML Data transformation Metabolite identification m_arpe_george_neg_metabolite_profiling_mass_spectrometry_v2_maf.tsv
CELL_Glc13_25mM_Normo_15 Extraction none Chromatography Agilent 1290 Infinity UHPLC ACQUITY UPLC BEH C18 (1.7 µm, 2.1 mm × 150 mm; Waters) reverse phase C13 Mass spectrometry negative 100-1500 Agilent 6550 iFunnel Q-TOF electrospray ionization quadrupole time-of-flight CELL_Glc13_25mM_Normo_15 CELL_Glc13_25mM_Normo_15.mzXML Data transformation Metabolite identification m_arpe_george_neg_metabolite_profiling_mass_spectrometry_v2_maf.tsv
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List of study files   Subset

File
audit
metexplore_mapping.json
CELL_Glc12_25mM_Normo_18.mzXML
CELL_Glc13_25mM_Normo_14.mzXML
a_arpe_george_neg_metabolite_profiling_mass_spectrometry.txt
CELL_Glc12_25mM_Normo_17.mzXML
CELL_Glc13_25mM_Normo_13.mzXML
CELL_Glc13_05mM_Normo_03.mzXML
CELL_Glc12_05mM_Normo_06.mzXML
CELL_Glc12_05mM_Normo_05.mzXML
CELL_Glc12_25mM_Normo_16.mzXML
s_ARPE_GeoRge_NEG.txt
CELL_Glc13_05mM_Normo_01.mzXML
m_arpe_george_neg_metabolite_profiling_mass_spectrometry_v2_maf.tsv
CELL_Glc13_05mM_Normo_02.mzXML
CELL_Glc13_25mM_Normo_15.mzXML
CELL_Glc12_05mM_Normo_04.mzXML
i_Investigation.txt

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MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)