Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA219038

Local Sample ID:	IL05_U_12_LCQTOFpos
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

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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.