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.

Study ID	ST002285
Study Title	Multiplatform mass spectrometry-based analysis of Leishmania donovani infected macrophages at different time points after infection
Study Type	Mutiplatform mass spectrometry-based metabolomics, time course experiment
Study Summary	The 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
Department	Chemistry and Biochemistry
Laboratory	Centre for Metabolomics and Bioanalysis (CEMBIO)
Last Name	Fernández García
First Name	Miguel
Address	Centro de Metabolómica y Bioanálisis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, 28660 Boadilla del Monte. España
Email	mig.fernandez.ce@ceindo.ceu.es
Phone	+0034690090778
Submit Date	2022-07-01
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2024-07-01
Release Version	1

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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.

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.