Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA136547

Local Sample ID:	Rothia35_03
Subject ID:	SU001685
Subject Type:	Bacteria
Subject Species:	Rothia mucilaginosa
Taxonomy ID:	43675
Genotype Strain:	RmFLR01

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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
Rothia35_03	SA136547	FL017492	Ambient	Condition
Rothia35_03	SA136547	FL017492	4h	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.

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.

Sampleprep ID:

SP001691

Sampleprep Summary:

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