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MB Sample ID: SA136693
Local Sample ID: | Rothia6_02 |
Subject ID: | SU001685 |
Subject Type: | Bacteria |
Subject Species: | Rothia mucilaginosa |
Taxonomy ID: | 43675 |
Genotype Strain: | RmFLR01 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU001685 |
Subject Type: | Bacteria |
Subject Species: | Rothia mucilaginosa |
Taxonomy ID: | 43675 |
Genotype Strain: | RmFLR01 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Rothia6_02 | SA136693 | FL017496 | Anaerobic | Condition |
Rothia6_02 | SA136693 | FL017496 | 8h | Collection time |
Collection:
Collection ID: | CO001678 |
Collection Summary: | A quality control mixture of 29 unlabeled metabolite standards (Supporting information Table SI 1) was prepared to reach a final concentration of 1 mg/mL as stock solution. 50 µL aliquot from each stock solution was combined into a new tube and dried down. Then, the mixture was diluted to reach a final concentration of 50 µg/mL working solution. Rothia mucilaginosa strain RmFLR01 was isolated from a cystic fibrosis (CF) patient at the UC San Diego Adult CF Clinic. R. mucilaginosa cultures were grown in triplicates in artificial-sputum medium spiked with 100 mM [U-13C6] d-glucose (Cambridge Isotope Laboratory, Tewksbury, MA, USA) under anaerobic and aerobic conditions (5% CO2) at 37°C and harvested at 4, 8, 12 and 24 h for isotope tracer analyses. |
Sample Type: | Bacterial cells |
Treatment:
Treatment ID: | TR001698 |
Treatment Summary: | For the 29 unlabeled standards mixture, 10 µL of the final solution was dried down for GC-MS measurement. Derivatization and data acquisition of mixture aliquots by GC-MS were reproduced on three days for inter-day precision. Dried mixtures were derivatized by adding 10 µL of 40 mg/mL methoxyamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in pyridine (Sigma-Aldrich, St. Louis, MO, USA) and shaking at 30°C for 1.5 h. Subsequently, 90 µL MTBSTFA (Sigma-Aldrich, St. Louis, MO, USA) was added with 13 fatty acid methyl esters (FAMEs) as retention index markers and shaken at 80°C for 30 min. Samples were immediately transferred to crimp top vials and injected onto each GC-MS instrument. Same samples of R. mucilaginosa cultures were extracted using published methods [19]. Samples were added 1 mL pre-chilled, degassed acetonitrile: isopropanol: water (v/v/v 3:3:2, Fisher Scientific) followed by vortexing 30 s and shaking at 4°C for 5 min. Samples were centrifuged for 2 min at 12,210 × g to precipitate debris from extracts. Supernatants were collected and split into two equal portions. One aliquot was dried to completeness in a Labconco cold trap centrifuge evaporator and then resuspended in 0.5 mL degassed acetonitrile: water (v/v 1:1, Fisher Scientific) to remove triacylglycerides. Resuspension solutions were vortexed for 30s and centrifuged for 2 min. Supernatants were transferred into clean Eppendorf tubes and dried down completely. Dried extracts were derivatized as given above. |
Sample Preparation:
Sampleprep ID: | SP001691 |
Sampleprep Summary: | For the 29 unlabeled standards mixture, 10 µL of the final solution was dried down for GC-MS measurement. Derivatization and data acquisition of mixture aliquots by GC-MS were reproduced on three days for inter-day precision. Dried mixtures were derivatized by adding 10 µL of 40 mg/mL methoxyamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in pyridine (Sigma-Aldrich, St. Louis, MO, USA) and shaking at 30°C for 1.5 h. Subsequently, 90 µL MTBSTFA (Sigma-Aldrich, St. Louis, MO, USA) was added with 13 fatty acid methyl esters (FAMEs) as retention index markers and shaken at 80°C for 30 min. Samples were immediately transferred to crimp top vials and injected onto each GC-MS instrument. Same samples of R. mucilaginosa cultures were extracted using published methods [19]. Samples were added 1 mL pre-chilled, degassed acetonitrile: isopropanol: water (v/v/v 3:3:2, Fisher Scientific) followed by vortexing 30 s and shaking at 4°C for 5 min. Samples were centrifuged for 2 min at 12,210 × g to precipitate debris from extracts. Supernatants were collected and split into two equal portions. One aliquot was dried to completeness in a Labconco cold trap centrifuge evaporator and then resuspended in 0.5 mL degassed acetonitrile: water (v/v 1:1, Fisher Scientific) to remove triacylglycerides. Resuspension solutions were vortexed for 30s and centrifuged for 2 min. Supernatants were transferred into clean Eppendorf tubes and dried down completely. Dried extracts were derivatized as given above. |
Combined analysis:
Analysis ID | AN002641 | AN002642 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | GC | GC |
Chromatography system | Agilent 7890A | Agilent 7890A |
Column | Restek Rtx-5Sil (30m x 0.25mm,0.25um) | Restek Rtx-5Sil (30m x 0.25mm,0.25um) |
MS Type | EI | EI |
MS instrument type | QTOF | Single quadrupole |
MS instrument name | Agilent 7200 QTOF | Agilent 5977 |
Ion Mode | POSITIVE | POSITIVE |
Units | peak height | peak height |
Chromatography:
Chromatography ID: | CH001951 |
Chromatography Summary: | an Agilent 7890 GC system installed with a Restek RTX-5Sil MS column (30m length, 0.25 mm i.d, 0.25 μM df, 95% dimethyl/5%diphenyl polysiloxane film) with an additional 10m guard column. For Q-TOF-MS analyses,GC parameters were used by injecting 1 µL of derivatized sample into the GC in splitless mode at an injection temperature of 250°C and a constant flow of 1 mL/min. The initial oven temperature was held at 60°C for 0.5 min, and ramped at a rate of 10°C/min to 325°C that was maintained for 10 min for a total run time of 37 min. |
Instrument Name: | Agilent 7890A |
Column Name: | Restek Rtx-5Sil (30m x 0.25mm,0.25um) |
Chromatography Type: | GC |
MS:
MS ID: | MS002453 |
Analysis ID: | AN002641 |
Instrument Name: | Agilent 7200 QTOF |
Instrument Type: | QTOF |
MS Type: | EI |
MS Comments: | MassHunter Quantitative Analysis B.07.00 version was used to process the data |
Ion Mode: | POSITIVE |
MS ID: | MS002454 |
Analysis ID: | AN002642 |
Instrument Name: | Agilent 5977 |
Instrument Type: | Single quadrupole |
MS Type: | EI |
MS Comments: | MassHunter Quantitative Analysis B.07.00 version was used to process the data |
Ion Mode: | POSITIVE |