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MB Sample ID: SA331560
| Local Sample ID: | Blank_1 |
| Subject ID: | SU003168 |
| Subject Type: | Bacteria |
| Subject Species: | Staphylococcus aureus |
| Taxonomy ID: | 1280 |
| Genotype Strain: | ATCC 43300 |
| Age Or Age Range: | NA |
| Weight Or Weight Range: | NA |
| Height Or Height Range: | NA |
| Gender: | Not applicable |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU003168 |
| Subject Type: | Bacteria |
| Subject Species: | Staphylococcus aureus |
| Taxonomy ID: | 1280 |
| Genotype Strain: | ATCC 43300 |
| Age Or Age Range: | NA |
| Weight Or Weight Range: | NA |
| Height Or Height Range: | NA |
| Gender: | Not applicable |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Blank_1 | SA331560 | FL039207 | Extraction blank | treatment |
Collection:
| Collection ID: | CO003161 |
| Collection Summary: | An untargeted metabolomics study was carried out to explore the mechanism(s) of action of C12-o-(BG-D)-10 against MRSA ATCC 43300 using a concentration of 48 µg/mL (i.e. 3×MIC). Samples were taken and analyzed at the 1-, 3-, and 6-h time points in 4 biological replicates. An overnight culture was prepared by inoculating a single colony into 100 mL CAMHB in 250 mL conical flasks (Pyrex) and incubating the suspension in a shaker at 37°C and 180 rpm for ~16 h. After incubation overnight, log-phase cells were prepared in fresh MHB and then incubated for 2 h at 37°C at 180 rpm to log phase with a starting bacterial inoculum of 108 CFU/mL. Then, C12-o-(BG-D)-10 was added to obtain the desired concentration of 48 µg/mL (3 x MIC), in parallel to a CLO-free control for each replicate. The flasks were then incubated at 37°C with shaking at 180 rpm. At each time point (0, 1, 3, and 6 h), 15-mL samples were transferred to 50-mL Falcon tubes for quenching, and the optical density reading at 600 nm (OD600) was then measured and normalized to the pre-treatment level of approximately ~0.5 with fresh CAMHB. Samples were then centrifuged at 3,220 g and 4°C for 10 min, and the supernatants were removed. The pellets were stored at -80°C until metabolite extraction. The experiment was performed in 4 biological replicates to reduce the bias from inherent random variation. |
| Sample Type: | Bacterial cells |
Treatment:
| Treatment ID: | TR003177 |
| Treatment Summary: | An untargeted metabolomics study was carried out to explore the mechanism(s) of action of C12-o-(BG-D)-10 against MRSA ATCC 43300 using a concentration of 48 µg/mL (i.e. 3×MIC). Samples were taken and analyzed at the 1-, 3-, and 6-h time points in 4 biological replicates. An overnight culture was prepared by inoculating a single colony into 100 mL CAMHB in 250 mL conical flasks (Pyrex) and incubating the suspension in a shaker at 37°C and 180 rpm for ~16 h. After incubation overnight, log-phase cells were prepared in fresh MHB and then incubated for 2 h at 37°C at 180 rpm to log phase with a starting bacterial inoculum of 108 CFU/mL. Then, C12-o-(BG-D)-10 was added to obtain the desired concentration of 48 µg/mL (3 x MIC), in parallel to a CLO-free control for each replicate. The flasks were then incubated at 37°C with shaking at 180 rpm. At each time point (0, 1, 3, and 6 h), 15-mL samples were transferred to 50-mL Falcon tubes for quenching, and the optical density reading at 600 nm (OD600) was then measured and normalized to the pre-treatment level of approximately ~0.5 with fresh CAMHB. Samples were then centrifuged at 3,220 g and 4°C for 10 min, and the supernatants were removed. The pellets were stored at -80°C until metabolite extraction. The experiment was performed in 4 biological replicates to reduce the bias from inherent random variation. |
| Treatment Dose: | 48 ug/ml |
| Cell Media: | CAMHB |
Sample Preparation:
| Sampleprep ID: | SP003174 |
| Sampleprep Summary: | The bacterial pellets were washed twice in 1 mL of 0.9% saline and then centrifuged at 3,220 g and 4°C for 5 min to remove residual extracellular metabolites and medium components. The washed pellets were resuspended in a cold extraction solvent (chloroform-methanol-water at 1:3:1, vol/vol) containing 1 µM each of the internal standards 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), piperazine-N, N-bis (2-ethanesulfonic acid) (PIPES), and Tris. The samples were then frozen in liquid nitrogen, thawed on ice, and vortexed to release the intracellular metabolites (3 times). Next, the samples were transferred to 1.5-mL Eppendorf tubes and centrifuged at 14,000 g at 4°C for 10 min to remove any particulate matter. Finally, 200 µL of the supernatant was transferred into injection vials for liquid chromatography-mass spectrometry (LC-MS) analysis. An equal volume of each sample was combined and used as a quality control (QC) sample |
Combined analysis:
| Analysis ID | AN005006 | AN005007 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
| Column | SeQuant ZIC-HILIC (150 x 4.6mm,3.5um) | SeQuant ZIC-HILIC (150 x 4.6mm,3.5um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak height | peak height |
Chromatography:
| Chromatography ID: | CH003782 |
| Chromatography Summary: | pHILIC using basic buffer pH9 |
| Methods Filename: | Metabolomics_pHILIC_Parkville_v1.pdf |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | SeQuant ZIC-HILIC (150 x 4.6mm,3.5um) |
| Column Pressure: | 60 bar at starting conditions. 180 bar at %A |
| Column Temperature: | 25 C |
| Flow Gradient: | 0 min - 80%B, 15 min - 50%B, 18 min - 5%B, 21 min - 5%B, 24 min - 80%B, 32 min - 80%B |
| Flow Rate: | 0.3 ml/min |
| Injection Temperature: | 4 C |
| Internal Standard: | CAPS, CHAPS, PIPES |
| Solvent A: | 100% water; 20 mM ammonium carbonate |
| Solvent B: | 100% acetonitrile |
| Analytical Time: | 32 min |
| Capillary Voltage: | 3.5 kV |
| Oven Temperature: | 25 C |
| Washing Buffer: | syringe wash 50% IPA |
| Weak Wash Solvent Name: | 50% IPA |
| Strong Wash Volume: | 50% IPA |
| Sample Loop Size: | 25 uL |
| Sample Syringe Size: | 25 uL |
| Randomization Order: | yes |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004746 |
| Analysis ID: | AN005006 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | acquired in full scan using polarity switching in positive and negative ion detection modes |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 300 C |
| Capillary Voltage: | 4 kV |
| Collision Energy: | NA |
| Collision Gas: | NA |
| Dry Gas Flow: | 50 |
| Dry Gas Temp: | 120 |
| Ion Source Temperature: | 120 C |
| Ionization: | ESI |
| Mass Accuracy: | 3 ppm |
| Precursor Type: | [M+H]+ |
| Source Temperature: | 300 |
| Acquisition Parameters File: | Metabolomics_pHILIC_Parkville_v1.pdf |
| Analysis Protocol File: | PQMS3-MPMF-WIN-0501_LCMS_data_acquisition_for_untargeted_metabolomics_analysis.pdf |
| MS ID: | MS004747 |
| Analysis ID: | AN005007 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | acquired in full scan using polarity switching in positive and negative ion detection modes |
| Ion Mode: | NEGATIVE |
| Capillary Temperature: | 300 C |
| Capillary Voltage: | 3.5 kV |
| Collision Energy: | NA |
| Collision Gas: | NA |
| Dry Gas Flow: | 50 |
| Dry Gas Temp: | 120 |
| Ion Source Temperature: | 120 C |
| Ionization: | ESI |
| Mass Accuracy: | 3 ppm |
| Precursor Type: | [M-H]- |
| Source Temperature: | 300 |
| Acquisition Parameters File: | Metabolomics_pHILIC_Parkville_v1.pdf |
| Analysis Protocol File: | PQMS3-MPMF-WIN-0501_LCMS_data_acquisition_for_untargeted_metabolomics_analysis.pdf |
