Return to study ST001864 main page
MB Sample ID: SA174472
| Local Sample ID: | 84 |
| Subject ID: | SU001941 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001941 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 84 | SA174472 | FL021438 | POMSF, 1h | group |
Collection:
| Collection ID: | CO001934 |
| Collection Summary: | Banked blood (Saint Louis Children’s Hospital) and fresh blood [healthy donors] was washed and stored at 50% hematocrit at 4ºC up to 1 month past the clinical expiration date and collection date, respectively. Parasite strains provided by the Malaria Research and Reference Reagent Resource Center (MR4) as follows: 3D7 (MRA-102); K1 (MRA-159); D10 (MRA-201); and IPC-5202 (MRA-1240). Unless indicated, erythrocytes were cultured as previously described at 2% hematocrit in complete media (incomplete media supplemented with 27mM sodium bicarbonate, 11mM glucose, 5mM HEPES, 1mM sodium pyruvate, 0.37mM hypoxanthine, 0.01mM thymidine, 10ug/mL gentamicin, and 0.5% Albumax (Thermo-Fisher) under 5% O2/5% CO2/90% N2 atmosphere at 37ºC (71). |
| Sample Type: | Blood (whole) |
Treatment:
| Treatment ID: | TR001953 |
| Treatment Summary: | Freshly collected erythrocytes were washed, resuspended to 10% hematocrit in buffer [25mM HEPES (pH 7.4), 120mM NaCl, 5.4mM KCl, 1.8mM CaCl2, and 1mM NaH2PO4], incubated for 1 hour at 37ºC, then centrifuged at 2000xg for 5 minutes at 4ºC, resuspended in fresh wash buffer to 30-40% hematocrit, and split into 210uL aliquots of packed erythrocytes. Erythrocytes were resuspended in 253uL of RPMI containing 11.9mM D-[1,2,3-13C] glucose (Millipore-Sigma) and either treated with POM-SF or POM-HEX at five times the EC50s for MetHb formation, or an untreated solvent control with the balance volume to 550uL using washing buffer. All samples were incubated at 37ºC shaking at 500 RPM. Triplicate samples of supernatants and packed erythrocytes were collected at 0, 0.5, 1, and 6-hour intervals and snap-frozen in liquid N2. |
Sample Preparation:
| Sampleprep ID: | SP001947 |
| Sampleprep Summary: | RBC and media samples were extracted at 1:10 and 1:20 dilutions, respectively, in ice-cold 5:3:2 MeOH:MeCN:H2O v/v/v, with vigorous vortexing at 4 degrees C for 30 min. The extraction buffer was supplemented with 40uM 3-13C lactate to allow absolute quantification of lactate isotopologues, including lactate generated via glycolysis ([1,2,3-13C3] enriched) vs. the pentose phosphate pathway ([2,3-13C2] enriched). Supernatants were clarified via centrifugation (10,000 g, 10 min, 4 C) then analyzed by UHPLC-MS. |
Combined analysis:
| Analysis ID | AN003021 | AN003022 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Vanquish | Vanquish |
| Column | Kinetex XB-C18 (150 x 2.1mm,1.7um) | Kinetex XB-C18 (150 x 2.1mm,1.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | NEGATIVE | POSITIVE |
| Units | peak area | peak area |
Chromatography:
| Chromatography ID: | CH002239 |
| Chromatography Summary: | 10 μl of RBC extracts were injected and separated through a 3 min isocratic elution on a Kinetex XB-C18 column (150 × 2.1 mm i.d., 1.7 μm particle size – Phenomenex, Torrance, CA, USA) at 250 μl/min (mobile phase: 5% acetonitrile, 95% 18 mΩ H2O, 0.1% formic acid; column temperature: 25°C). |
| Instrument Name: | Vanquish |
| Column Name: | Kinetex XB-C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 25 |
| Flow Gradient: | isocratic |
| Flow Rate: | 250 ul/min |
| Solvent A: | 5% acetonitrile/95%water; 0.1% formic acid |
| Solvent B: | isocratic |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002810 |
| Analysis ID: | AN003021 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The UHPLC system was coupled online with a QExactive mass spectrometer (Thermo, San Jose, CA, USA), scanning in Full MS mode (2 μscans) at 70,000 resolution from 60-900 m/z, with 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in positive ion mode. Calibration was performed before each analysis using a positive calibration mix (Piercenet – Thermo Fisher, Rockford, IL, USA). Limits of detection (LOD) were characterized by determining the smallest injected amino acid amount required to provide a signal to noise (S/N) ratio greater than three using < 5 ppm error on the accurate intact mass. Based on a conservative definition for Limit of Quantitation (LOQ), these values were calculated to be three fold higher than determined LODs. MS data acquired from the QExactive was converted from .raw file format to.mzXML format using MassMatrix (Cleveland, OH, USA). Amino acid assignments were performed using MAVEN (Princeton, NJ, USA). The MAVEN software platform provides tools for peak picking, feature detection and metabolite assignment against the KEGG pathway database. Assignments were further confirmed using a process for chemical formula determination using isotopic patterns and accurate intact mass (Clasquin et al. 2012). Analyte retention times were confirmed by comparison with external standard retention times, as indicated above. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS002811 |
| Analysis ID: | AN003022 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The UHPLC system was coupled online with a QExactive mass spectrometer (Thermo, San Jose, CA, USA), scanning in Full MS mode (2 μscans) at 70,000 resolution from 60-900 m/z, with 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in positive ion mode. Calibration was performed before each analysis using a positive calibration mix (Piercenet – Thermo Fisher, Rockford, IL, USA). Limits of detection (LOD) were characterized by determining the smallest injected amino acid amount required to provide a signal to noise (S/N) ratio greater than three using < 5 ppm error on the accurate intact mass. Based on a conservative definition for Limit of Quantitation (LOQ), these values were calculated to be three fold higher than determined LODs. MS data acquired from the QExactive was converted from .raw file format to.mzXML format using MassMatrix (Cleveland, OH, USA). Amino acid assignments were performed using MAVEN (Princeton, NJ, USA). The MAVEN software platform provides tools for peak picking, feature detection and metabolite assignment against the KEGG pathway database. Assignments were further confirmed using a process for chemical formula determination using isotopic patterns and accurate intact mass (Clasquin et al. 2012). Analyte retention times were confirmed by comparison with external standard retention times, as indicated above. |
| Ion Mode: | POSITIVE |
