Summary of Study ST002285

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 PR001465. The data can be accessed directly via it's Project DOI: 10.21228/M8C12D 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.

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Study IDST002285
Study TitleMultiplatform mass spectrometry-based analysis of Leishmania donovani infected macrophages at different time points after infection
Study TypeMutiplatform mass spectrometry-based metabolomics, time course experiment
Study SummaryThe project aims to measure targeted and non-targeted metabolite data of intracellular extracts of uninfected and Leishmania-donovani infected macrophages at 0, 12, 36 and 72 hours post infection using a multiplatform mass spectrometry approach combining CE-TOF/MS (polar metabolites), LC-QTOF/MS (non-polar metabolites) and LC-QqQ/MS (polar metabolites) to characterize the dynamics of metabolic alterations ocurring in the human macrophage upon L. donovani infection.
Institute
Universidad CEU San Pablo
DepartmentChemistry and Biochemistry
LaboratoryCentre for Metabolomics and Bioanalysis (CEMBIO)
Last NameFernández García
First NameMiguel
AddressCentro de Metabolómica y Bioanálisis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, 28660 Boadilla del Monte. España
Emailmig.fernandez.ce@ceindo.ceu.es
Phone+0034690090778
Submit Date2022-07-01
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2024-07-01
Release Version1
Miguel Fernández García Miguel Fernández García
https://dx.doi.org/10.21228/M8C12D
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001465
Project DOI:doi: 10.21228/M8C12D
Project Title:Immunometabolic Networks in the Regulation of Visceral Leishmaniasis
Project Summary:Despite a wealth of information about the mechanisms underlying immunity to disease following Leishmania infection, the correlates of protection in human leishmaniasis remain poorly defined. In that sense, a complete understanding of Leishmania biology and host-pathogen interactions is mandatory to fully recognize the molecular and cellular foundations of leishmaniasis pathogenesis. Recent studies have been demonstrating how innate cell metabolism associates to the development of resistance towards infection. As the causality in the immunometabolic connections remains untested, we propose to employ an integrative approach aiming at mapping the Leishmania-induced shifts in metabolic networks in macrophages and neutrophils, deciphering the molecular mechanisms of the metabolic shifts and establishing its causal relationship with the anti-leishmanial immune response mediated by these two types of cells. Dissecting these aspects is critical not only for an in-depth understanding of the biological mechanisms underlying Leishmania pathogenesis, but also to drive significant advances in the development of novel diagnostic and immunotherapeutic approaches.
Institute:University of Minho
Department:School of Medicine
Laboratory:Life and Health Sciences Research Institute (ICVS)
Last Name:Fernandez
First Name:Miguel
Address:Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho
Email:mig.fernandez.ce@ceindo.ceu.es
Phone:690090778

Subject:

Subject ID:SU002371
Subject Type:Cultured cells
Subject Species:Homo sapiens + Leishmania donovani
Taxonomy ID:9606 and 5661
Genotype Strain:Human: PBMCs from 7 human donors. L. donovani: MHOM/IN/82/Patra1 and MHOM/ET/1967/Hu3

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens + Leishmania donovani (Factor headings shown in green)

mb_sample_id local_sample_id Infection (Factor) Time (Factor)
SA218847IL07_2_12_LCQTOFneginfected 12
SA218848IL01_1_12_LCQQQneginfected 12
SA218849IL03_1_12_LCQQQneginfected 12
SA218850IL06_2_12_LCQTOFneginfected 12
SA218851IL01_1_12_LCQTOFneginfected 12
SA218852IL05_2_12_LCQTOFneginfected 12
SA218853IL02_2_12_LCQTOFneginfected 12
SA218854IL03_2_12_LCQTOFneginfected 12
SA218855IL04_2_12_LCQTOFneginfected 12
SA218856IL04_1_12_LCQQQneginfected 12
SA218857IL05_1_12_LCQQQneginfected 12
SA218858IL04_2_12_CETOFinfected 12
SA218859IL03_2_12_CETOFinfected 12
SA218860IL02_2_12_CETOFinfected 12
SA218861IL05_2_12_CETOFinfected 12
SA218862IL06_2_12_CETOFinfected 12
SA218863IL06_1_12_LCQQQneginfected 12
SA218864IL07_1_12_LCQQQneginfected 12
SA218865IL07_2_12_CETOFinfected 12
SA218866IL01_2_12_LCQTOFneginfected 12
SA218867IL02_1_12_LCQTOFposinfected 12
SA218868IL07_1_12_LCQTOFneginfected 12
SA218869IL07_2_12_LCQTOFposinfected 12
SA218870IL06_2_12_LCQTOFposinfected 12
SA218871IL05_2_12_LCQTOFposinfected 12
SA218872IL06_1_12_LCQTOFneginfected 12
SA218873IL05_1_12_LCQTOFneginfected 12
SA218874IL02_1_12_LCQTOFneginfected 12
SA218875IL03_1_12_LCQTOFneginfected 12
SA218876IL04_1_12_LCQTOFneginfected 12
SA218877IL04_2_12_LCQTOFposinfected 12
SA218878IL03_2_12_LCQTOFposinfected 12
SA218879IL04_1_12_LCQTOFposinfected 12
SA218880IL03_1_12_LCQTOFposinfected 12
SA218881IL01_2_12_CETOFinfected 12
SA218882IL05_1_12_LCQTOFposinfected 12
SA218883IL06_1_12_LCQTOFposinfected 12
SA218884IL02_2_12_LCQTOFposinfected 12
SA218885IL01_2_12_LCQTOFposinfected 12
SA218886IL07_1_12_LCQTOFposinfected 12
SA218887IL01_1_12_LCQTOFposinfected 12
SA218888IL02_1_12_LCQQQneginfected 12
SA218889IL01_1_12_CETOFinfected 12
SA218890IL06_1_12_CETOFinfected 12
SA218891IL02_2_12_LCQQQneginfected 12
SA218892IL03_2_12_LCQQQneginfected 12
SA218893IL04_2_12_LCQQQneginfected 12
SA218894IL05_1_12_CETOFinfected 12
SA218895IL01_2_12_LCQQQneginfected 12
SA218896IL02_1_12_CETOFinfected 12
SA218897IL03_1_12_CETOFinfected 12
SA218898IL04_1_12_CETOFinfected 12
SA218899IL07_2_12_LCQQQneginfected 12
SA218900IL05_2_12_LCQQQneginfected 12
SA218901IL07_1_12_CETOFinfected 12
SA218902IL06_2_12_LCQQQneginfected 12
SA218903IL04_2_36_LCQTOFposinfected 36
SA218904IL03_2_36_LCQTOFposinfected 36
SA218905IL02_2_36_LCQTOFposinfected 36
SA218906IL05_2_36_LCQTOFposinfected 36
SA218907IL02_1_36_LCQQQneginfected 36
SA218908IL06_2_36_LCQTOFposinfected 36
SA218909IL02_1_36_LCQTOFneginfected 36
SA218910IL04_1_36_LCQTOFneginfected 36
SA218911IL05_1_36_LCQTOFneginfected 36
SA218912IL07_2_36_LCQQQneginfected 36
SA218913IL06_2_36_LCQQQneginfected 36
SA218914IL07_1_36_LCQTOFposinfected 36
SA218915IL06_1_36_LCQTOFposinfected 36
SA218916IL05_1_36_LCQTOFposinfected 36
SA218917IL04_1_36_LCQTOFposinfected 36
SA218918IL02_2_36_LCQQQneginfected 36
SA218919IL07_1_36_LCQTOFneginfected 36
SA218920IL05_2_36_LCQQQneginfected 36
SA218921IL04_2_36_LCQQQneginfected 36
SA218922IL06_1_36_LCQTOFneginfected 36
SA218923IL02_1_36_LCQTOFposinfected 36
SA218924IL02_2_36_LCQTOFneginfected 36
SA218925IL05_2_36_CETOFinfected 36
SA218926IL04_2_36_CETOFinfected 36
SA218927IL06_2_36_CETOFinfected 36
SA218928IL07_2_36_CETOFinfected 36
SA218929IL06_1_36_CETOFinfected 36
SA218930IL03_2_36_CETOFinfected 36
SA218931IL02_2_36_CETOFinfected 36
SA218932IL05_1_36_LCQQQneginfected 36
SA218933IL04_1_36_LCQQQneginfected 36
SA218934IL06_1_36_LCQQQneginfected 36
SA218935IL07_1_36_LCQQQneginfected 36
SA218936IL07_1_36_CETOFinfected 36
SA218937IL03_2_36_LCQTOFneginfected 36
SA218938IL05_1_36_CETOFinfected 36
SA218939IL07_2_36_LCQTOFneginfected 36
SA218940IL04_1_36_CETOFinfected 36
SA218941IL07_2_36_LCQTOFposinfected 36
SA218942IL06_2_36_LCQTOFneginfected 36
SA218943IL05_2_36_LCQTOFneginfected 36
SA218944IL02_1_36_CETOFinfected 36
SA218945IL04_2_36_LCQTOFneginfected 36
SA218946IL07_2_72_LCQQQneginfected 72
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Collection:

Collection ID:CO002364
Collection Summary:Cloned lines of Leishmania donovani (MHOM/IN/82/Patra1 and MHOM/ET/1967/Hu3) were maintained with weekly subpassages at 27 ºC in complete RPMI 1640 medium, supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin plus 100 mg/mL streptomycin and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. Only parasites under ten passages were used in the experimental work. Peripheral blood mononuclear cells (PBMCs) were enriched from blood from healthy volunteers using a density gradient with Histopaque 1077. The mononuclear phase was recovered and washed with PBS. Next, cells were labeled with magnetic CD14 MicroBeads and CD14+ monocytes were positively separated using an MS column. Human CD14+ monocytes were differentiated in macrophages using recombinant macrophage colony stimulating factor 1 (M-CSF). Briefly, monocytes were plated at 1·106 cells/mL with 20 ng/mL of M-CSF. Growth factor was renewed at day 4 post-differentiation. The macrophages were used 7 days after differentiation. Macrophages were infected with L. donovani promastigotes at a 1:10 ratio. After 4 hours of incubation, non-phagocytosed parasites were removed, and cells were recovered. Macrophages were left in culture for 12, 36 and 72 h for metabolomic analysis. Uninfected macrophages were used as a control. To isolate uninfected and L. donovani-infected human monocyte-derived macrophages for further metabolomic analyses, cells were recovered, washed with phosphate buffer saline (PBS) and transferred into a previously weighted Eppendorf. After centrifugation the supernatant was discarded, and the pellet was immediately frozen in liquid nitrogen and stored at -80 ºC.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR002383
Treatment Summary:Macrophages were infected with L. donovani promastigotes at a 1:10 ratio. After 4 hours of incubation, non-phagocytosed parasites were removed, and cells were recovered. Macrophages were left in culture for 12, 36 and 72 h for metabolomic analysis (HMDMLd+). Uninfected macrophages were used as a control (HMDMLd-).

Sample Preparation:

Sampleprep ID:SP002377
Sampleprep Summary:To enhance the metabolome coverage of the samples, a double extraction was performed based on a previously described methodology 15. Briefly, 500 µL of cold methanol were added to L. donovani-infected and uninfected macrophage pellets. Subsequently, 170 mg of of 425-600 µm acid-washed glass beads (Sigma-Aldrich, Steinheim, Germany) were added to each sample. Samples were lysed using 4 subsequent cycles of bead-assisted lysis using a Tissuelyser® (t = 10 min, f = 50 Hz) followed by cooling in an ice bath (t = 10 min). To ensure a correct cellular lysis, the resulting cellular extracts were observed under Giemsa staining before and after cell lysis. Once lysis was performed, samples were kept under ice to ensure a correct extraction of the metabolites (t = 10 min). After centrifugation (12,000 × g, T = 4 °C, t = 10 min), 100 µL of the methanolic extract were separated for the analysis of metabolites by reversed-phase LC-QTOF/MS. To perform an increased extraction of polar metabolites, 140 µL of Milli-Q water were added to the initial extract. After gentle vortexing (t = 5 min), metabolite extraction on ice bath (t = 10 min) and centrifugation (12,000 × g, T = 4 °C, t = 10 min), the resulting volume of the MeOH/H2O extracts was aliquoted for both LC-QqQ/MS and CE-TOF/MS analyses. CEMS: CE-TOF/MS sample buffer solution was prepared by dissolving methionine sulfone (internal standard, Sigma-Aldrich, Steinheim, Germany) in Milli-Q water containing 0.1 M formic acid (Sigma-Aldrich, Steinheim, Germany) up to a 0.2 mM concentration. Samples containing 165 μL of H2O/MeOH HMDMLd+ and HMDMLd- metabolite extracts were evaporated to dryness under high vacuum. The dried samples were resuspended in 70 μL of CE-TOF/MS sample buffer solution by energic vortexing for 1 min. After subsequent centrifugation (12,600 × g, T = 4 ºC, t = 15 min), the resulting clear solution was analyzed by CE-TOF/MS. LC-QTOF/MS: Samples were prepared with 70 μL of the methanolic HMDMLd+ and HMDMLd- metabolite extracts. LC-QqQ/MS: A solution of 50.09 µM p-chlorophenylalanine (Sigma-Aldrich, Steinheim, Germany) in Milli-Q water was used as LC-QqQ/MS internal standard solution. A mixture of 15 µL of LC-QqQ/MS internal standard solution and 200 µL of the MeOH/H2O HMDMLd+ and HMDMLd- metabolite extracts was evaporated under high vacuum in a SpeedVac® concentrator (T = 40 °C; t = 2 h). The resulting evaporated extract was resuspended in 50 µL of H2O. Vials were centrifuged (3,000 × g, T = 4 °C, t = 10 min) prior to injection.

Combined analysis:

Analysis ID AN003734 AN003735 AN003736 AN003737
Analysis type MS MS MS MS
Chromatography type CE Reversed phase Reversed phase Reversed phase
Chromatography system Agilent 7100 CE Agilent 1200 Agilent 1200 Agilent 1290 Infinity
Column bare-fused silica capillary (96 cm,50um),Agilent Technologies Agilent Zorbax Extend C18 RRHT (50 x 2.1mm,1.8 um) Agilent Zorbax Extend C18 RRHT (50 x 2.1mm,1.8 um) Agilent Zorbax RRHD SB-C18 (100 x 2.1mm,1.8um)
MS Type ESI ESI ESI ESI
MS instrument type TOF QTOF QTOF Triple quadrupole
MS instrument name Agilent 6224 TOF Agilent 6545 QTOF Agilent 6545 QTOF Agilent 6460 QQQ
Ion Mode POSITIVE POSITIVE NEGATIVE NEGATIVE
Units Abundances Abundances Abundances Normalized abundances

Chromatography:

Chromatography ID:CH002766
Chromatography Summary:CE separation for charged metabolites which ionice in positive mode, in acid media.
Instrument Name:Agilent 7100 CE
Column Name:bare-fused silica capillary (96 cm,50um),Agilent Technologies
Column Temperature:20 ºC
Injection Temperature:20 ºC
Internal Standard:methionine sulfone
Sample Injection:50 mbar for 100 s hydrodynamical injection stacking of 100 mbar for 20 s
Capillary Voltage:30 kV
Running Buffer:1M formic acid in MeOH:H2O 1:9 (v/v)
Sheath Liquid:100 mL of Milli-Q water with 100 mL of 100% methanol (Thermo Fisher Scientific, Loughborough, UK), 4 µL of concentrated formic acid, 10 µL of 5 mM purine and 10 µL 2.5 hexakis (1H,1H,3H-tetrafluoropropoxy)phosphazene HP722 (CE-TOF/MS reference masses, Agilent Technologies, CA, USA)
Chromatography Type:CE
  
Chromatography ID:CH002767
Chromatography Summary:RPLC method for separating phospholipids and non very non-polar lipids
Instrument Name:Agilent 1200
Column Name:Agilent Zorbax Extend C18 RRHT (50 x 2.1mm,1.8 um)
Column Temperature:60 ºC
Flow Gradient:Initial conditions at time 0 were 5% B, held until 1 min. Next, the percentage of organic phase was gradually increased up to 80% B at 7 min and subsequently until 100% B at 11.5 min. The conditions were then returned to the starting conditions by 12 min, followed by a 3 min re-equilibration time. The total run time of the method was 15 min.
Flow Rate:0.6 mL/min
Injection Temperature:4 ºC
Sample Injection:8 uL
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH002768
Chromatography Summary:Adapted from Agilent dMRM database and method with minor modifications
Instrument Name:Agilent 1290 Infinity
Column Name:Agilent Zorbax RRHD SB-C18 (100 x 2.1mm,1.8um)
Column Temperature:35 ºC
Flow Gradient:Binary pump gradient: Time (min) flow (mL/min) %B; Quaternary pump gradient: Time (min) flow (mL/min) %C %D 0 0 1 99 23.95 0 1 99 24 0.2 1 99 27 0.2 1 99 27.5 0.3 1 99 43.35 0.3 1 99 43.5 0.3 1 99 52.25 0.2 100 0 59 0.2 100 0 59.9 0.2 1 99 60 0 1 99 0.00 0.25 0 2.5 0.25 0 7.5 0.25 20 13 0.25 45 20 0.25 99 24 0.25 99 24.05 0 99 24.1 0 0 58.95 0 0 59 0.20 0 60 0.25 0;
Flow Rate:0.25 mL/min
Injection Temperature:4 ºC
Sample Injection:20 uL
Solvent A:97% water/3% methanol; 15 mM acetic acid; 10 mM tributylamine
Solvent B:100% methanol; 15 mM acetic acid; 10 mM tributylamine;
Chromatography Type:Reversed phase

MS:

MS ID:MS003482
Analysis ID:AN003734
Instrument Name:Agilent 6224 TOF
Instrument Type:TOF
MS Type:ESI
MS Comments:mass range mz 68 to 1000 mass correction mz 121.0509 and 922.0098 mass calibrators Acq mode full scan AMH Profinder B.08.00 for feature deconvolution CMM Batch Search and in house library of standards with RMT
Ion Mode:POSITIVE
Capillary Voltage:3500 V
Dry Gas Flow:10 L/min
Dry Gas Temp:200 ºC
Fragment Voltage:125 V
Nebulizer:10 psi
Octpole Voltage:750 V
  
MS ID:MS003483
Analysis ID:AN003735
Instrument Name:Agilent 6545 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:full MS: mass range mz 50 to 3000 mass correction mz 121.0509 and 922.0098 mass calibrators iterative MSMS of pooled samples isolation width = 1.3 Da CE with slope and offset of 3.8 and 4.6, respectively AMH Profinder B.08.00 for feature deconvolution CMM Batch Search and in house library of standards with RMT AMH LipidAnnotator for in silico anotation of MSMS spectra
Ion Mode:POSITIVE
Capillary Voltage:4000 V
Dry Gas Flow:12 L/min
Dry Gas Temp:250 ºC
Fragment Voltage:125 V
Nebulizer:52 psi
Octpole Voltage:750 V
  
MS ID:MS003484
Analysis ID:AN003736
Instrument Name:Agilent 6545 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:full MS: mass range mz 50 to 3000 mass correction mz 121.0509 and 922.0098 mass calibrators iterative MSMS of pooled samples isolation width = 1.3 Da CE with slope and offset of 3.8 and 4.6, respectively AMH Profinder B.08.00 for feature deconvolution CMM Batch Search and in house library of standards with RMT AMH LipidAnnotator for in silico anotation of MSMS spectra
Ion Mode:NEGATIVE
Capillary Voltage:4000 V
Dry Gas Flow:12 L/min
Dry Gas Temp:250 ºC
Fragment Voltage:125 V
Nebulizer:52 psi
Octpole Voltage:750 V
  
MS ID:MS003485
Analysis ID:AN003737
Instrument Name:Agilent 6460 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:MRM isolation width narrow 1.3 Da AMH Quantitative Analysis (Quant My Way) for targeted data integration cycle time = 650 ms MRM repeats = 3 nozzle voltage = 500 V
Ion Mode:NEGATIVE
Capillary Voltage:2000 V
Dry Gas Flow:12 L/min
Dry Gas Temp:325 ºC
Nebulizer:45 psi
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