Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA274789

Local Sample ID:	2''-01-8398
Subject ID:	SU002837
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male

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Subject:

Subject ID:	SU002837
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
2''-01-8398	SA274789	FL035584	MGB4	Group

Collection:

Collection ID:	CO002830
Collection Summary:	Human prostate cancer cells LNCaP were grown in Roswell Park Memorial Institute (RPMI) 1640 Medium (Sigma Aldrich, Germany) supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, Germany). A combination of 1 % penicillin and streptomycin antibiotics was added to RPMI/FBS growth media. Cells were maintained in humidified incubator with conditions of 37◦C temperature and 5 % CO2.
Sample Type:	LNCaP cells
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002846
Treatment Summary:	Three biological replicates were prepared for each treatment condition in T75 cm2 ﬂasks (MGB4 and/or Doxorubicin and control group), and each replicate was seeded with approximately 2 × 106 LNCaP cells, then separately treated with MGB4 (2.15 μM) and/or Doxorubicin (1.18 μM) and incubated for 24 h. A uniform number of cells was seeded in T75 cm2 ﬂask for each sample to avoid the effect of variable cell number on the outcome of the experiment. After incubation, cells were washed twice with phosphate-buffered saline solution (PBS) and then collected by trypsinization. Finally, cells were collected as pellets by centrifugation at 1200 rounds per minute (rpm) for 10 min at room temperature and resuspended in 1 mL 1× PBS for further analysis. Cells were maintained in the same conditions during incubation, and all samples were collected simultaneously (Figure 5).

Treatment ID:

TR002846

Treatment Summary:

Three biological replicates were prepared for each treatment condition in T75 cm2 ﬂasks (MGB4 and/or Doxorubicin and control group), and each replicate was seeded with approximately 2 × 106 LNCaP cells, then separately treated with MGB4 (2.15 μM) and/or Doxorubicin (1.18 μM) and incubated for 24 h. A uniform number of cells was seeded in T75 cm2 ﬂask for each sample to avoid the effect of variable cell number on the outcome of the experiment. After incubation, cells were washed twice with phosphate-buffered saline solution (PBS) and then collected by trypsinization. Finally, cells were collected as pellets by centrifugation at 1200 rounds per minute (rpm) for 10 min at room temperature and resuspended in 1 mL 1× PBS for further analysis. Cells were maintained in the same conditions during incubation, and all samples were collected simultaneously (Figure 5).

Sample Preparation:

Sampleprep ID:	SP002843
Sampleprep Summary:	we used chloroform/methanol extraction protocol to increase the coverage of the extracted metabolites. At first, the samples (cells and buffer) were transferred into Eppendorf tubes then centrifuged at 14000 rpm for 5 min. Afterward, the buffer was discarded, and the cells were preserved. To each sample, 400 µL of the mixture containing one protease inhibitor tablet and 10 mL of lysis buffer was added. Following rest for 10 minutes, samples were transferred to 10 mL tubes, vortexed for 2–4 minutes, and sonicated with a COPLEY probe-sonicator (QSONICA SONICATOR, USA) for 30 seconds while utilizing a 30 % amplifier in an ice bath. The samples were then transferred to Eppendorf tubes and centrifuged for 5 minutes at 14000 rpm. The supernatant was then transferred to another Eppendorf, and 400 µL of methanol and 300 µL of chloroform were added. Following that, the samples were vortexed for 30 seconds and centrifuged for 5 minutes at 14000 rpm. After that, two metabolite-containing layers are obtained, after transferring the upper layer of each sample to glass vials, 400 µl of methanol was added, followed by vertexing and centrifugation. The remaining supernatant was transferred to the same glass vials used before for the drying step, with the remaining protein pellets being air-dried for proteomics. A dried metabolomics sample was resuspended in 200 µL (0.1% formic acid in water) and injected into HPLC to be analysed by Q-TOF MS.

Sampleprep ID:

SP002843

Sampleprep Summary:

we used chloroform/methanol extraction protocol to increase the coverage of the extracted metabolites. At first, the samples (cells and buffer) were transferred into Eppendorf tubes then centrifuged at 14000 rpm for 5 min. Afterward, the buffer was discarded, and the cells were preserved. To each sample, 400 µL of the mixture containing one protease inhibitor tablet and 10 mL of lysis buffer was added. Following rest for 10 minutes, samples were transferred to 10 mL tubes, vortexed for 2–4 minutes, and sonicated with a COPLEY probe-sonicator (QSONICA SONICATOR, USA) for 30 seconds while utilizing a 30 % amplifier in an ice bath. The samples were then transferred to Eppendorf tubes and centrifuged for 5 minutes at 14000 rpm. The supernatant was then transferred to another Eppendorf, and 400 µL of methanol and 300 µL of chloroform were added. Following that, the samples were vortexed for 30 seconds and centrifuged for 5 minutes at 14000 rpm. After that, two metabolite-containing layers are obtained, after transferring the upper layer of each sample to glass vials, 400 µl of methanol was added, followed by vertexing and centrifugation. The remaining supernatant was transferred to the same glass vials used before for the drying step, with the remaining protein pellets being air-dried for proteomics. A dried metabolomics sample was resuspended in 200 µL (0.1% formic acid in water) and injected into HPLC to be analysed by Q-TOF MS.

Combined analysis:

Analysis ID	AN004428
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Bruker Elute
Column	Hamilton Intensity Solo 2 C18 (100 x 2.1 mm, 1.8 um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Bruker timsTOF
Ion Mode	POSITIVE
Units	AU

Chromatography:

Chromatography ID:	CH003327
Chromatography Summary:	Samples were chromatographically separated by inline reversed-phase chromatography using the Elute HPG 1300 pumps and Elute Autosampler (Bruker, Darmstadt, Germany) with solvent A 0.1% FA in HPLC grade water and solvent B 0.1% FA in ACN. A Hamilton Intensity Solo 2 C18 column (100 mm x 2.1 mm, 1.8µm beads) was maintained at 35C. For metabolomics, 10 µL was injected twice for each sample and eluted using a 30-minute gradient as follows: 1% ACN was held for 2 minutes, ramping to 99% ACN over 15 minutes, held at 99% ACN for 3 minutes before re-equilibrating to 1% ACN for 10 minutes. Flow rates were 250 µL/min for elution and 350 µL/min for re-equilibration.
Instrument Name:	Bruker Elute
Column Name:	Hamilton Intensity Solo 2 C18 (100 x 2.1 mm, 1.8 um)
Column Temperature:	35
Flow Gradient:	1%B to 99%B in 15 min
Flow Rate:	250 uL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004175
Analysis ID:	AN004428
Instrument Name:	Bruker timsTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The MS analysis was performed using a TimsTOF (Bruker, Darmstadt, Germany) with Apollo II electrospray ionization (ESI) source. The drying gas was set to flow at 10 L/min and the drying temperature to 220C and the nebulizer pressure to 2.2 bar. The capillary voltage was 4500 V and the end plate offset 500V. For metabolomics the scan range was 20-1300 m/z. The collision energy was set to 20 eV, the cycle time to 0.5 seconds with a relative minimum intensity threshold of 400 counts per thousand and target intensity of 20,000. Sodium formate was injected as an external calibrant in the first 0.3 minutes of each LC-MS/MS run. MetaboScape 4.0 software was used for metabolite processing and statistical analysis (Bruker Daltonics). The following parameters for molecular feature identification and "bucketing" were set in the T-ReX 2D/3D workflow: For peak detection, a minimum intensity threshold of 1,000 counts is required, as well as a minimum peak duration of 7 spectra, with feature quantification determine using peak area. The file masses were recalibrated based on the external calibrant injected between 0-0.3 min.
Ion Mode:	POSITIVE