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MB Sample ID: SA218880
| Local Sample ID: | IL03_1_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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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:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| IL03_1_12_LCQTOFpos | SA218880 | FL026521 | infected | Infection (Factor) |
| IL03_1_12_LCQTOFpos | SA218880 | FL026521 | 12 | Time (Factor) |
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 |
