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MTBLS630:  Genome-scale metabolic modelling of responses to polymyxins in Pseudomonas aeruginosa

 Authors: Yan Zhu , Jian Li , Meiling Han , Mohd Hafidz Mahamad Maifiah

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  Submitted: 16-Feb-2018 , Release date: 28-Feb-2018 , Update date: 28-Feb-2018

 Submitted by:  Yan Zhu  |   Study status: Public

Study Description

Background: Pseudomonas aeruginosa often causes multidrug-resistant infections in immunocompromised patients and polymyxins are often used as the last-line therapy. Alarmingly, resistance to polymyxins has been increasingly reported worldwide recently. To rescue this last-resort class of antibiotics, it is necessary to systematically understand how P. aeruginosa alters its metabolism in response to polymyxin treatment, thereby facilitating the development of effective therapies. To this end, a genome-scale metabolic model (GSMM) was employed to analyse bacterial metabolic changes at the systems level. Findings: A high-quality GSMM iPAO1 was constructed for P. aeruginosa PAO1 for antimicrobial pharmacological research. Model iPAO1 encompasses an additional periplasmic compartment and contains 3,022 metabolites, 4,265 reactions and 1,458 genes in total. Growth prediction on 190 carbon and 95 nitrogen sources achieved an accuracy of 89.1%, outperforming all reported P. aeruginosa models. Notably, prediction of the essential genes for growth achieved a high accuracy of 87.9%. Metabolic simulation showed that lipid A modifications associated with polymyxin resistance exert a limited impact on bacterial growth and metabolism, but remarkably change the physiochemical properties of the outer membrane. Modelling with transcriptomics constraints revealed a broad range of metabolic responses to polymyxin treatment, including reduced biomass synthesis, upregulated amino acids catabolism, induced flux through the tricarboxylic acid cycle, and increased redox turnover. Conclusions: Overall, iPAO1 represents the most comprehensive GSMM constructed to date for Pseudomonas. It provides a powerful systems pharmacology platform for the elucidation of complex killing mechanisms of antibiotics.

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  Organism(s)

Pseudomonas aeruginosa

  Study Design Description

CHEBI:polymyxin

untargeted metabolites

CSEO:Lipidomics

genome-scale metabolic model

CHMO:high-performance liquid chromatography-mass spectrometry

  Experimental Factors

Strain

Time elapsed

Presence of polymyxin

Replicate

Protocol Description
Sample collection P. aeruginosa PAK was obtained from Moskowitz Laboratory (Massachusetts General Hospital, MA, USA). The broth microdilution MICs of PAK was 1 mg/l. Prior to experiments, PAK was subcultured onto nutrient agar plates and incubated for 16-18 hr at 37 °C. A single colony was then inoculated into 10 ml of cation-adjusted Mueller-Hinton broth (CaMHB, Oxoid) and incubated for overnight at 37 °C with shaking at 150 rpm. The overnight culture was diluted by 1:100 into 5 different reservoirs with 100 ml fresh CaMHB media and then grown to an optical density at 600 nm (OD600) of 0.50 (~10^8 CFU/ml). The bacterial cultures of PAK was then treated with polymyxin B at 4 mg/l for 1 and 4 hr. The untreated bacterial cultures served as a control sample. Control and treatment samples (n=3) at 4 hr were collected for lipid A profiling; whereas only control samples (n=5) at 1 hr were collected for lipidomics study. To ensure the bacterial cell counts were comparable at each time point, the bacterial cultures were normalized according to OD600 of 0.50.

The culture of P. aeruginosa PAO1 was prepared on a nutrient agar plate from the frozen stock (-80 °C) and incubated for 16-18 hr at 37 °C. For the overnight culture, a colony of P. aeruginosa PAO1 was inoculated into 15 ml CaMHB and incubated for 16-18 hr at 37 °C with shaking at 150 rpm. The overnight culture was diluted with 1:100 into 4 different reservoirs of 200 ml fresh CaMHB. To obtain enough cells for the metabolomics experiment, the culture was grown to an optical density at 600 nm (OD600) of ~0.5 (~10^8 CFU/ml) and subject to polymyxin B (1 mg/l) treatment for 1 hr. Untreated bacterial culture served as a control sample.
Extraction Lipid A profiling for PAK:
Lipid A was isolated by mild acid hydrolysis. In brief, the bacterial cell pellets were harvested from 100 ml of normalized culture (OD600 = 0.50) via centrifugation at 3,220 x g for 20 min and washed twice with 5 ml PBS. The cell pellets were then re-suspended in 4 ml PBS, and followed by 5 ml chloroform and 10 ml methanol to make a single-phase Bligh-Dyer (chloroform/methanol/water, 1:2:0.8, v/v). After 15-min centrifugation at 3,220 x g, the supernatant was removed and left LPS in the pellets. After one washing with 5 ml single-phase Bligh-Dyer solvent, the LPS pellets were re-suspended in 10.8 ml of hydrolysis buffer (50 mM sodium acetate pH 4.5 with 1% sodium dodecyl sulfate [SDS]) and homogenized via sonication through a probe tip sonicator (Misonix, USA) at a constant duty cycle (20 s at 50% output). The samples were then incubated in a boiling water bath for 45 min and allowed to cool to room temperature. To extract lipids after hydrolysis, 12 ml chloroform and 12 ml methanol were added to the 10.8 ml hydrolysis solution to make a double-phase Bligh-Dyer (chloroform/methanol/water, 1:1:0.9, v/v). The lower phase containing lipid A was finally collected and dried under nitrogen gas stream. Structural analysis of lipid A was performed using mass spectrometry in negative ion mode on a Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer.

Lipidomics analysis for PAK:
For lipidomics study, cellular metabolites of PAK were extracted by a previously optimized method with slight modifications. Briefly, the untreated samples were collected at 1 hr for metabolite extraction. For the fingerprint samples (i.e. intracellular metabolites), 20 ml of the bacterial culture was collected and immediately transferred into a 50 ml ice-cold falcon tube. The samples were then shortly quenched in a dry ice/ethanol bath for ~30 s to stop the metabolic processes. Samples were then normalized according to OD600 at 0.50 to ensure the bacterial cell counts are at ~10^8 CFU/ml. The cell pellets were then collected after centrifugation at 3,220 x g at 4 °C for 10 min, while the supernatant was collected for extracellular metabolites (i.e. footprint). After washing twice with 2 ml ice-cold 0.9% NaCl, the cell pellets were then resuspended in 500 µl chloroform/methanol/water (CMW, 1:3:1, v/v) containing 1 µM generic internal standards (CHAPS, CAPS, PIPES and TRIS). A triple freeze-thaw process was followed to lyse the cells and release cellular metabolites. The extracted samples were centrifuged at 3,220 x g at 4 °C for 10 min and 300 µl supernatant was collected, which is followed by a further centrifugation at 14,000 x g for 10 min at 4 °C to obtain particle-free supernatants (200 µl) for LC-MS analysis. For footprint samples, 10 µl of the filtered supernatant was mixed with 250 µl of CMW (1:3:1, v/v) solution and then centrifuged at 14,000 x g for 10 min at 4 °C to collect 200 µl particle-free supernatant for LC-MS analysis.

Metabolomics analysis for PAO1:
Cellular metabolites of P. aeruginosa PAO1 were extracted by the previously optimized method with slight modifications. Samples were collected 1 hr from untreated and polymyxin treatment (1 mg/l) groups for metabolite extraction and viable counting. For the fingerprint samples (i.e. intracellular metabolites), 20 ml of the bacterial culture was collected and immediately transferred onto the ice. Quickly, all the samples were quenched in a dry ice/ethanol bath and preserved on ice for all following steps. Samples were normalized by optical density (OD600 nm) of ~0.5 (~10^8 CFU/ml). The samples then were centrifuged for 10 min at 3,220 x g at 4 °C. The cell pellets were washed 3 times with 0.9% NaCl (4 °C) and centrifuged for 3 min at 3,220 x g at 4 °C. Cellular metabolites were extracted with chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) (total volume of 250 µl) containing generic internal standards (CHAPS, CAPS, PIPES and TRIS) at 1 µM. Samples were frozen in liquid nitrogen and allowed to thaw on ice, and the freeze-thaw process was repeated 3 times to lyse the cells and release cellular metabolites. The extracted samples were centrifuged for 10 min at 3,220 x g at 4 °C and the supernatant was collected and further centrifuged at 14,000 x g for 10 min at 4 °C. The final supernatant samples (200 µl) were collected into the injector vial for LC-MS analysis.
Chromatography Lipid A profiling for PAK:
Structural and semiquantitative analysis of lipid A was performed on a Dionex U3000 high-performance liquid chromatography system (HPLC) coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher). The Phenomenex Synergi Hydro-RP 80 Å column (50 x 2 mm, 4 µm) was maintained at 40 °C, while the samples were stored at 4 °C. Lipid A samples in chloroform/methanol/isopropanol/water (1:1:1:0.5, v/v) were eluted using a gradient that consisted of 40% isopropanol (IPA) and 60% Milli-Q water with 8 mM ammonium formate and 2 mM formic acid as mobile phase A and 98% IPA and 2% Milli-Q water with 8 mM ammonium formate and 2 mM formic acid as mobile phase B. The flow rate was 0.2 ml/min within the first 15 min and increased to 0.5 ml/min from 16 to 22 min. The gradient started with 70% mobile phase A and 30% mobile phase B, followed by a linear gradient to a final composition of 100% mobile phase B, which was maintained for 4 min. A 3 min reequilibration of the column with 70% mobile phase A was performed prior to the next injection.

Lipidomics analysis for PAK:
Lipidomic analysis was conducted on a Dionex U3000 high-performance liquid chromatography system (HPLC) coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher). The Ascentis Express C8 column (50 mm x 2.1 mm, 2.7 µm, Sigma-Aldrich, 53831-U) was maintained at 40 °C and the samples were controlled at 4 °C. The flow rate was 0.2 ml/min from 0 to 24 min, then increased to 0.5 ml/min from 25 to 30 min. For lipidomics studies, the linear gradient starting from 100% mobile phase A (40% of isopropanol and 60% of Milli-Q water with 8 mM ammonium formate and 2 mM formic acid) to a final composition of 35% mobile phase A and 65% mobile phase B (98% of isopropanol and 2% of Milli-Q water with 8 mM ammonium formate and 2 mM formic acid) was employed.

Metabolomics analysis for PAO1:
Samples were analysed on a Dionex high-performance liquid chromatograph (U3000 RSLC HPLC, Thermo Fisher) coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher) with a ZIC-pHILIC column (5 µm, polymeric, 150 x 4.6 mm; SeQuant, Merck). The LC solvent consisted of 20 mM ammonium carbonate (A) and acetonitrile (B) with a multi-step gradient system from 80% B to 50% B over 15 min, then to 5% B at 18 min, followed by a wash with 5% B for 3 min, and re-equilibration for 8 min with 80% B at a flow rate of 0.3 ml/min.
Mass Spectrometry Lipid A profiling for PAK:
Structural and semiquantitative analysis of lipid A was performed on a Dionex U3000 high-performance liquid chromatography system (HPLC) in tandem with a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher) in negative mode with a resolution at 70,000. The mass range was from 167 to 2500 m/z. The electrospray voltage was set as 3.50 kV, and nitrogen was used as collision gas.

Lipidomics analysis for PAK:
Lipidomic analysis was conducted on a Dionex U3000 high-performance liquid chromatography system (HPLC) in tandem with a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher) in both positive and negative mode with a resolution at 35,000. The mass scanning range was from 167 to 2,000 m/z. The electrospray voltage was set as 3.50 kV and nitrogen was used as collision gas.

Metabolomics analysis for PAO1:
Samples were analysed on a Dionex high-performance liquid chromatograph (U3000 RSLC HPLC, Thermo Fisher) in tandem with a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher) in both positive and negative electro-spray ionization (ESI) mode (rapid switching) with a resolution at 35,000. The mass scanning range was from 85 to 1,275 m/z.

The injection sample volume was 10 µl and the run time was 32 min. All samples were analysed in the same run and the chromatographic peaks, signal reproducibility and analyte stability were monitored by assessment of pooled biological quality control (PBQC) samples (aliquot of 10 µl of each sample) analysed periodically throughout the batch, internal standards and total ion chromatograms for each sample. Mixtures of pure standards containing over 200 metabolites were analysed within the batch to aid in the identification of metabolites.
Data transformation Global metabolomics analyses were performed using mzMatch and IDEOM (http://mzmatch.sourceforge.net/ideom.php) free software. Raw LC-MS data were converted to mzXML format and chromatogram peaks were detected using XCMS66 and saved in the peakML format.
Metabolite identification The program Mzmatch.R was used to align samples and filter peaks based on minimum detectable intensity (100000), reproducibility (relative standard deviation (RSD) for all replicates <0.5) and peak shape (codadw >0.8). Mzmatch.R was also used to retrieve LC-MS peak intensities for missing peaks and for the annotation of related peaks. Unwanted noise and artefact peaks were eliminated using IDEOM with default parameters. Metabolites were putatively identified by the exact mass within 2 ppm, after correction for loss or gain of a proton in negative and positive ESI mode, respectively. Retention time was employed to confirm the identification of each metabolite based on the available authentic standards. Putative identification of other metabolites was determined using exact mass and predicted retention time based on the Kyoto Encyclopedia of Genes and Genomes (KEGG), MetaCyc and LIPIDMAPS databases, with preference given to bacterial metabolites annotated in EcoCyc. Quantification of each metabolite was calculated using the raw peak height and is expressed relative to the average peak height for their paired susceptible strain.
Source Name Organism Variant Organism part Protocol REF Sample Name Strain Time elapsed Unit Presence of polymyxin Replicate
PAK control 4h rep 1 Pseudomonas aeruginosa Pseudomonas aeruginosa str. PAK whole organism Sample collection LA_PAK_1_C PAK 4 hour No 1
PAK pmbB 4h rep 1 Pseudomonas aeruginosa Pseudomonas aeruginosa str. PAK whole organism Sample collection LA_PAK_1_P PAK 4 hour Yes 1
PAK control 4h rep 2 Pseudomonas aeruginosa Pseudomonas aeruginosa str. PAK whole organism Sample collection LA_PAK_2_C PAK 4 hour No 2
PAK pmbB 4h rep 2 Pseudomonas aeruginosa Pseudomonas aeruginosa str. PAK whole organism Sample collection LA_PAK_2_P PAK 4 hour Yes 2
PAK control 4h rep 3 Pseudomonas aeruginosa Pseudomonas aeruginosa str. PAK whole organism Sample collection LA_PAK_3_C PAK 4 hour No 3
PAK pmbB 4h rep 3 Pseudomonas aeruginosa Pseudomonas aeruginosa str. PAK whole organism Sample collection LA_PAK_3_P PAK 4 hour Yes 3
PAK 1h rep1 Pseudomonas aeruginosa Pseudomonas aeruginosa str. PAK whole organism Sample collection PAK_1h_1 PAK 1 hour No 1
PAK 1h rep2 Pseudomonas aeruginosa Pseudomonas aeruginosa str. PAK whole organism Sample collection PAK_1h_2 PAK 1 hour No 2
PAK 1h rep3 Pseudomonas aeruginosa Pseudomonas aeruginosa str. PAK whole organism Sample collection PAK_1h_3 PAK 1 hour No 3
PAK 1h rep4 Pseudomonas aeruginosa Pseudomonas aeruginosa str. PAK whole organism Sample collection PAK_1h_4 PAK 1 hour No 4
PAK 1h rep5 Pseudomonas aeruginosa Pseudomonas aeruginosa str. PAK whole organism Sample collection PAK_1h_5 PAK 1 hour No 5
PAO1 control 0h rep1 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_C_0h_3 PAO1 0 hour No 3
PAO1 control 0h rep2 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_C_0h_4 PAO2 0 hour No 4
PAO1 control 0h rep3 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_C_0h_5 PAO3 0 hour No 5
PAO1 control 0h rep4 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_C_0h_6 PAO4 0 hour No 6
PAO1 control 1h rep1 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_C_1h_3 PAO5 1 hour No 3
PAO1 control 1h rep2 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_C_1h_4 PAO6 1 hour No 4
PAO1 control 1h rep3 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_C_1h_5 PAO7 1 hour No 5
PAO1 control 1h rep4 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_C_1h_6 PAO8 1 hour No 6
PAO1 polymyxin B 0h rep1 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_PB_0h_3 PAO9 0 hour Yes 3
PAO1 polymyxin B 0h rep2 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_PB_0h_4 PAO10 0 hour Yes 4
PAO1 polymyxin B 0h rep3 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_PB_0h_5 PAO11 0 hour Yes 5
PAO1 polymyxin B 0h rep4 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_PB_0h_6 PAO12 0 hour Yes 6
PAO1 polymyxin B 1h rep1 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_PB_1h_3 PAO13 1 hour Yes 3
PAO1 polymyxin B 1h rep2 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_PB_1h_4 PAO14 1 hour Yes 4
PAO1 polymyxin B 1h rep3 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_PB_1h_5 PAO15 1 hour Yes 5
PAO1 polymyxin B 1h rep4 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 whole organism Sample collection PAO1_PB_1h_6 PAO16 1 hour Yes 6
Validations marked with (*) have been allowed by the MetaboLights Curators.
Click here for the detailed description of Validations.
Condition Status Description Requirement Group Message
PASSES Study Title MANDATORY STUDY OK
PASSES Study Description MANDATORY STUDY OK
PASSES Study text successfully parsed OPTIONAL STUDY OK
PASSES Study Contact(s) have listed email MANDATORY CONTACT OK
PASSES Sample(s) MANDATORY SAMPLES OK
PASSES Sample Name consistency check MANDATORY ASSAYS OK
PASSES Publication(s) associated with this Study MANDATORY PUBLICATION OK
PASSES Minimal Experimental protocol MANDATORY PROTOCOLS OK
PASSES Comprehensive Experimental protocol OPTIONAL PROTOCOLS OK
PASSES Extraction protocol description MANDATORY PROTOCOLS OK
PASSES Data transformation protocol description MANDATORY PROTOCOLS OK
PASSES Metabolite Identification protocol description MANDATORY PROTOCOLS OK
PASSES Mass spectrometry protocol description MANDATORY PROTOCOLS OK
PASSES Chromatography protocol description MANDATORY PROTOCOLS OK
PASSES Sample Collection protocol description MANDATORY PROTOCOLS OK
PASSES Protocols text successfully parsed OPTIONAL PROTOCOLS OK
PASSES Organism name MANDATORY ORGANISM OK
PASSES Organism part MANDATORY ORGANISM OK
PASSES Study Factors MANDATORY FACTORS OK
PASSES Assay platform information OPTIONAL ASSAYS OK
PASSES Assay has raw files referenced MANDATORY FILES OK
PASSES Assay referenced raw files detection in filesystem MANDATORY FILES OK
PASSES Raw files in the Assay(s) have the correct format MANDATORY FILES OK
PASSES Assay(s) MANDATORY ASSAYS OK
PASSES All Assays have Metabolite Assignment File (MAF) referenced OPTIONAL FILES OK
PASSES Metabolite Assignment File (MAF) is present in Study folder MANDATORY FILES OK
PASSES Metabolite Assignment File (MAF) has correct format MANDATORY FILES OK
PASSES Metabolite Identification File (MAF) content MANDATORY FILES OK
PASSES ISA-Tab investigation file check MANDATORY ISATAB OK

Assay  1

Assay file name: a_mtbls630_PAO1_C1_mass_spectrometry.txt
Technology: mass spectrometry
Platform: HPLC-LTQ-MS

Data

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
PAO1_C_1h_3 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap C1_3 C1_3.raw Data transformation C1_3.mzML Metabolite identification m_mtbls630_PAO1_C1_mass_spectrometry_v2_maf.tsv
PAO1_C_1h_4 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap C1_4 C1_4.raw Data transformation C1_4.mzML Metabolite identification m_mtbls630_PAO1_C1_mass_spectrometry_v2_maf.tsv
PAO1_C_1h_5 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap C1_5 C1_5.raw Data transformation C1_5.mzML Metabolite identification m_mtbls630_PAO1_C1_mass_spectrometry_v2_maf.tsv
PAO1_C_1h_6 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap C1_6 C1_6.raw Data transformation C1_6.mzML Metabolite identification m_mtbls630_PAO1_C1_mass_spectrometry_v2_maf.tsv

Assay  2

Assay file name: a_mtbls630_LA_mass_spectrometry.txt
Technology: mass spectrometry
Platform: HPLC-LTQ-MS

Data

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
LA_PAK_1_C Extraction chloroform/methanol/water, 1:1:0.9, v/v LA_PAK_1_C Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Synergi Hydro-RP 80 Å (4 µm, 2 mm x 50 mm; Phenomenex) reverse phase Mass spectrometry alternating 167-2500 Thermo Scientific Q Exactive electrospray ionization orbitrap LA_PAK_1_C 4_LA_PAK-1-4-C.raw Data transformation 4_LA_PAK-1-4-C.mzML Metabolite identification m_mtbls630_LA_mass_spectrometry_v2_maf.tsv
LA_PAK_1_P Extraction chloroform/methanol/water, 1:1:0.9, v/v LA_PAK_1_P Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Synergi Hydro-RP 80 Å (4 µm, 2 mm x 50 mm; Phenomenex) reverse phase Mass spectrometry alternating 167-2500 Thermo Scientific Q Exactive electrospray ionization orbitrap LA_PAK_1_P 5_LA_PAK-1-4-P.raw Data transformation 5_LA_PAK-1-4-P.mzML Metabolite identification m_mtbls630_LA_mass_spectrometry_v2_maf.tsv
LA_PAK_2_C Extraction chloroform/methanol/water, 1:1:0.9, v/v LA_PAK_2_C Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Synergi Hydro-RP 80 Å (4 µm, 2 mm x 50 mm; Phenomenex) reverse phase Mass spectrometry alternating 167-2500 Thermo Scientific Q Exactive electrospray ionization orbitrap LA_PAK_2_C 4_LA_PAK-2-4-C.raw Data transformation 4_LA_PAK-2-4-C.mzML Metabolite identification m_mtbls630_LA_mass_spectrometry_v2_maf.tsv
LA_PAK_2_P Extraction chloroform/methanol/water, 1:1:0.9, v/v LA_PAK_2_P Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Synergi Hydro-RP 80 Å (4 µm, 2 mm x 50 mm; Phenomenex) reverse phase Mass spectrometry alternating 167-2500 Thermo Scientific Q Exactive electrospray ionization orbitrap LA_PAK_2_P 5_LA_PAK-2-4-P.raw Data transformation 5_LA_PAK-2-4-P.mzML Metabolite identification m_mtbls630_LA_mass_spectrometry_v2_maf.tsv
LA_PAK_3_C Extraction chloroform/methanol/water, 1:1:0.9, v/v LA_PAK_3_C Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Synergi Hydro-RP 80 Å (4 µm, 2 mm x 50 mm; Phenomenex) reverse phase Mass spectrometry alternating 167-2500 Thermo Scientific Q Exactive electrospray ionization orbitrap LA_PAK_3_C 4_LA_PAK-3-4-C.raw Data transformation 4_LA_PAK-3-4-C.mzML Metabolite identification m_mtbls630_LA_mass_spectrometry_v2_maf.tsv
LA_PAK_3_P Extraction chloroform/methanol/water, 1:1:0.9, v/v LA_PAK_3_P Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Synergi Hydro-RP 80 Å (4 µm, 2 mm x 50 mm; Phenomenex) reverse phase Mass spectrometry alternating 167-2500 Thermo Scientific Q Exactive electrospray ionization orbitrap LA_PAK_3_P 5_LA_PAK-3-4-P.raw Data transformation 5_LA_PAK-3-4-P.mzML Metabolite identification m_mtbls630_LA_mass_spectrometry_v2_maf.tsv

Assay  3

Assay file name: a_mtbls630_PAO1_PB0_mass_spectrometry.txt
Technology: mass spectrometry
Platform: HPLC-LTQ-MS

Data

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
PAO1_PB_0h_3 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap PB0_3 PB0_3.raw Data transformation PB0_3.mzML Metabolite identification m_mtbls630_PAO1_PB0_mass_spectrometry_v2_maf.tsv
PAO1_PB_0h_4 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap PB0_4 PB0_4.raw Data transformation PB0_4.mzML Metabolite identification m_mtbls630_PAO1_PB0_mass_spectrometry_v2_maf.tsv
PAO1_PB_0h_5 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap PB0_5 PB0_5.raw Data transformation PB0_5.mzML Metabolite identification m_mtbls630_PAO1_PB0_mass_spectrometry_v2_maf.tsv
PAO1_PB_0h_6 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap PB0_6 PB0_6.raw Data transformation PB0_6.mzML Metabolite identification m_mtbls630_PAO1_PB0_mass_spectrometry_v2_maf.tsv

Assay  4

Assay file name: a_mtbls630_PAK_mass_spectrometry.txt
Technology: mass spectrometry
Platform: HPLC-LTQ-MS

Data

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
PAK_1h_1 Extraction chloroform/methanol/water (CMW, 1:3:1, v/v) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Ascentis Express C8 (2.7 µm, 2.1 mm x 50 mm; Sigma-Aldrich) reverse phase Mass spectrometry alternating 167-2000 Thermo Scientific Q Exactive electrospray ionization orbitrap PAK-1-1 PAK-1-1.raw Data transformation PAK-1-1.mzML Metabolite identification m_mtbls630_PAK_mass_spectrometry_v2_maf.tsv
PAK_1h_2 Extraction chloroform/methanol/water (CMW, 1:3:1, v/v) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Ascentis Express C8 (2.7 µm, 2.1 mm x 50 mm; Sigma-Aldrich) reverse phase Mass spectrometry alternating 167-2000 Thermo Scientific Q Exactive electrospray ionization orbitrap PAK-2-1 PAK-2-1.raw Data transformation PAK-2-1.mzML Metabolite identification m_mtbls630_PAK_mass_spectrometry_v2_maf.tsv
PAK_1h_3 Extraction chloroform/methanol/water (CMW, 1:3:1, v/v) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Ascentis Express C8 (2.7 µm, 2.1 mm x 50 mm; Sigma-Aldrich) reverse phase Mass spectrometry alternating 167-2000 Thermo Scientific Q Exactive electrospray ionization orbitrap PAK-3-1 PAK-3-1.raw Data transformation PAK-3-1.mzML Metabolite identification m_mtbls630_PAK_mass_spectrometry_v2_maf.tsv
PAK_1h_4 Extraction chloroform/methanol/water (CMW, 1:3:1, v/v) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Ascentis Express C8 (2.7 µm, 2.1 mm x 50 mm; Sigma-Aldrich) reverse phase Mass spectrometry alternating 167-2000 Thermo Scientific Q Exactive electrospray ionization orbitrap PAK-4-1 PAK-4-1.raw Data transformation PAK-4-1.mzML Metabolite identification m_mtbls630_PAK_mass_spectrometry_v2_maf.tsv
PAK_1h_5 Extraction chloroform/methanol/water (CMW, 1:3:1, v/v) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Ascentis Express C8 (2.7 µm, 2.1 mm x 50 mm; Sigma-Aldrich) reverse phase Mass spectrometry alternating 167-2000 Thermo Scientific Q Exactive electrospray ionization orbitrap PAK-5-1 PAK-5-1.raw Data transformation PAK-5-1.mzML Metabolite identification m_mtbls630_PAK_mass_spectrometry_v2_maf.tsv

Assay  5

Assay file name: a_mtbls630_PAO1_PB1_mass_spectrometry.txt
Technology: mass spectrometry
Platform: HPLC-LTQ-MS

Data

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
PAO1_PB_1h_3 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap PB1_3 PB1_3.raw Data transformation PB1_3.mzML Metabolite identification m_mtbls630_PAO1_PB1_mass_spectrometry_v2_maf.tsv
PAO1_PB_1h_4 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap PB1_4 PB1_4.raw Data transformation PB1_4.mzML Metabolite identification m_mtbls630_PAO1_PB1_mass_spectrometry_v2_maf.tsv
PAO1_PB_1h_5 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap PB1_5 PB1_5.raw Data transformation PB1_5.mzML Metabolite identification m_mtbls630_PAO1_PB1_mass_spectrometry_v2_maf.tsv
PAO1_PB_1h_6 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex Ultimate 3000 HPLC system Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap PB1_6 PB1_6.raw Data transformation PB1_6.mzML Metabolite identification m_mtbls630_PAO1_PB1_mass_spectrometry_v2_maf.tsv

Assay  6

Assay file name: a_mtbls630_PAO1_C0_mass_spectrometry.txt
Technology: mass spectrometry
Platform: HPLC-LTQ-MS

Data

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
PAO1_C_0h_3 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex UltiMate 3000 RSLC System ZIC-pHILIC (5 µm, 4.6 mm x 150 mm; Merck Sequant) HILIC Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap C0_3 C0_3.raw Data transformation C0_3.mzML Metabolite identification m_mtbls630_PAO1_C0_mass_spectrometry_v2_maf.tsv
PAO1_C_0h_4 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex UltiMate 3000 RSLC System ZIC-pHILIC (5 µm, 4.6 mm x 150 mm; Merck Sequant) HILIC Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap C0_4 C0_4.raw Data transformation C0_4.mzML Metabolite identification m_mtbls630_PAO1_C0_mass_spectrometry_v2_maf.tsv
PAO1_C_0h_5 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex UltiMate 3000 RSLC System ZIC-pHILIC (5 µm, 4.6 mm x 150 mm; Merck Sequant) HILIC Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap C0_5 C0_5.raw Data transformation C0_5.mzML Metabolite identification m_mtbls630_PAO1_C0_mass_spectrometry_v2_maf.tsv
PAO1_C_0h_6 Extraction chloroform:methanol:water (CMW; 1:3:1, v/v; -80 °C) Chromatography Thermo Scientific Dionex UltiMate 3000 RSLC System ZIC-pHILIC (5 µm, 4.6 mm x 150 mm; Merck Sequant) HILIC Mass spectrometry alternating 85-1275 Thermo Scientific Q Exactive electrospray ionization orbitrap C0_6 C0_6.raw Data transformation C0_6.mzML Metabolite identification m_mtbls630_PAO1_C0_mass_spectrometry_v2_maf.tsv

Pathways - Assay  1



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)

Pathways - Assay  2



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)

Pathways - Assay  3



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)

Pathways - Assay  4



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)

Pathways - Assay  5



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)

Pathways - Assay  6



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)
  Download study (FTP)  |    Download metadata    

Aspera Download Details:

List of study files   Subset

File
PAK-4-1.raw
i_Investigation.txt
PAK-1-1.raw
PAK-4-1.mzML
PAK-3-1.raw
PAK-2-1.raw
PAK-3-1.mzML
PAK-5-1.mzML
PAK-5-1.raw
PAK-2-1.mzML
PAK-1-1.mzML
PB1_6.mzML
C0_5.mzML
C0_6.raw
C1_3.mzML
PB1_3.raw
C0_5.raw
C1_4.mzML
PB0_3.raw
PB0_3.mzML
PB0_6.mzML
PB0_4.raw
C1_5.raw
PB1_5.mzML
C0_6.mzML
C1_4.raw
PB0_6.raw
PB0_5.mzML
PB1_4.mzML
C1_5.mzML
C0_4.raw
C0_3.mzML
C1_6.raw
PB1_6.raw
PB0_5.raw
C1_6.mzML
PB1_4.raw
PB1_3.mzML
C0_4.mzML
C1_3.raw
PB1_5.raw
PB0_4.mzML
lipidomics_PAK.txt
metabolomics_PAO1_C1.txt
4_LA_PAK-3-4-C.raw
C0_3.raw
5_LA_PAK-3-4-P.mzML
5_LA_PAK-3-4-P.raw
5_LA_PAK-1-4-P.mzML
4_LA_PAK-1-4-C.raw
5_LA_PAK-2-4-P.mzML
5_LA_PAK-1-4-P.raw
4_LA_PAK-2-4-C.raw
4_LA_PAK-2-4-C.mzML
audit
metabolomics_PAO1_C1.xlsx
lipidomics_PAK.xlsx
metabolomics_PAO1_PB0.xlsx
metabolomics_PAO1_PB1.xlsx
lipid_A_profiling_PAK.xlsx
5_LA_PAK-2-4-P.raw
metabolomics_PAO1_C0.xlsx
metabolomics_PAO1_C0.txt
4_LA_PAK-1-4-C.mzML
4_LA_PAK-3-4-C.mzML
metabolomics_PAO1_PB1.txt
lipid_A_profiling_PAK.txt
metexplore_mapping.json
metabolomics_PAO1_C0.tsv
metabolomics_PAO1_PB0.tsv
lipidomics_PAK.tsv
metabolomics_PAO1_C1.tsv
metabolomics_PAO1_PB0.txt
metabolomics_PAO1_PB1.tsv
metabolomics_PAO1_C0_unicode.txt
lipid_A_profiling_PAK_unicode.txt
lipidomics_PAK_unicode.txt
lipid_A_profiling_PAK.tsv
s_mtbls630.txt
m_mtbls630_PAO1_C1_mass_spectrometry_v2_maf.tsv
m_mtbls630_PAO1_C0_mass_spectrometry_v2_maf.tsv
m_mtbls630_PAK_mass_spectrometry_v2_maf.tsv
m_mtbls630_LA_mass_spectrometry_v2_maf.tsv
a_mtbls630_PAK_mass_spectrometry.txt
a_mtbls630_PAO1_PB0_mass_spectrometry.txt
a_mtbls630_PAO1_C0_mass_spectrometry.txt
a_mtbls630_LA_mass_spectrometry.txt
m_mtbls630_PAO1_PB1_mass_spectrometry_v2_maf.tsv
m_mtbls630_PAO1_PB0_mass_spectrometry_v2_maf.tsv
a_mtbls630_PAO1_C1_mass_spectrometry.txt
a_mtbls630_PAO1_PB1_mass_spectrometry.txt

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