Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA174355

Local Sample ID:	20190525_Indranil_pos_spme_meta1_2
Subject ID:	SU001938
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Female

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Subject:

Subject ID:	SU001938
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Female

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
20190525_Indranil_pos_spme_meta1_2	SA174355	FL021403	2	Replicate
20190525_Indranil_pos_spme_meta1_2	SA174355	FL021403	Control	Treatment
20190525_Indranil_pos_spme_meta1_2	SA174355	FL021403	0_day	Treatment

Collection:

Collection ID:	CO001931
Collection Summary:	Human breast epithelial MCF10A cells were kindly provided by Prof. Senthil Muthuswamy (Beth Israel Deaconess Medical Center, Harvard Medical School). Cells were cultured in DMEM/F-12 supplemented with 5% Horse serum, EGF 20 ng/mL (Sigma), Insulin 10 μg/mL (Sigma), Hydrocortisone 0.5 mg/mL (Sigma), Cholera toxin 100 ng/mL (Sigma), 100 units/mL Penicillin and 100 μg/mL Streptomycin (HyClone) and grown at 37C in a humidified incubator with 5% CO2. To induce EMT, cells were stimulated with 10 ng/mL TGF-β1 (Invivogen) and treatments were staggered such that all cells (plates) were harvested at the same time. To minimize cross-contamination (EV & Sec) and promiscuous background signaling (particularly for Phos), cells were cultured in serum-free conditions for 16 hours prior to harvesting. At the time of harvest, conditioned media were first transferred to fresh 50 mL tubes and kept on ice. Cells were washed once with ice-cold PBS and scraped off the plates in ice-cold PBS. Each sample was then distributed into multiple aliquots for multi-omics extractions, centrifuged at 800×g for 5 minutes at 4°C and stored as dry pellets at –80°C. Live cells were imaged in their culture vessels before harvesting using ZOE fluorescent cell imager (Bio-Rad).
Sample Type:	Breast cancer cells

Treatment:

Treatment ID:	TR001950
Treatment Summary:	Human breast epithelial MCF10A cells were kindly provided by Prof. Senthil Muthuswamy (Beth Israel Deaconess Medical Center, Harvard Medical School). Cells were cultured in DMEM/F-12 supplemented with 5% Horse serum, EGF 20 ng/mL (Sigma), Insulin 10 μg/mL (Sigma), Hydrocortisone 0.5 mg/mL (Sigma), Cholera toxin 100 ng/mL (Sigma), 100 units/mL Penicillin and 100 μg/mL Streptomycin (HyClone) and grown at 37°C in a humidified incubator with 5% CO2. To induce EMT, cells were stimulated with 10 ng/mL TGF-β1 (Invivogen) and treatments were staggered such that all cells (plates) were harvested at the same time. To minimize cross-contamination (EV & Sec) and promiscuous background signaling (particularly for Phos), cells were cultured in serum-free conditions for 16 hours prior to harvesting. At the time of harvest, conditioned media were first transferred to fresh 50 mL tubes and kept on ice. Cells were washed once with ice-cold PBS and scraped off the plates in ice-cold PBS. Each sample was then distributed into multiple aliquots for multi-omics extractions, centrifuged at 800×g for 5 minutes at 4°C and stored as dry pellets at –80°C. Live cells were imaged in their culture vessels before harvesting using ZOE fluorescent cell imager (Bio-Rad).

Treatment ID:

TR001950

Treatment Summary:

Human breast epithelial MCF10A cells were kindly provided by Prof. Senthil Muthuswamy (Beth Israel Deaconess Medical Center, Harvard Medical School). Cells were cultured in DMEM/F-12 supplemented with 5% Horse serum, EGF 20 ng/mL (Sigma), Insulin 10 μg/mL (Sigma), Hydrocortisone 0.5 mg/mL (Sigma), Cholera toxin 100 ng/mL (Sigma), 100 units/mL Penicillin and 100 μg/mL Streptomycin (HyClone) and grown at 37°C in a humidified incubator with 5% CO2. To induce EMT, cells were stimulated with 10 ng/mL TGF-β1 (Invivogen) and treatments were staggered such that all cells (plates) were harvested at the same time. To minimize cross-contamination (EV & Sec) and promiscuous background signaling (particularly for Phos), cells were cultured in serum-free conditions for 16 hours prior to harvesting. At the time of harvest, conditioned media were first transferred to fresh 50 mL tubes and kept on ice. Cells were washed once with ice-cold PBS and scraped off the plates in ice-cold PBS. Each sample was then distributed into multiple aliquots for multi-omics extractions, centrifuged at 800×g for 5 minutes at 4°C and stored as dry pellets at –80°C. Live cells were imaged in their culture vessels before harvesting using ZOE fluorescent cell imager (Bio-Rad).

Sample Preparation:

Sampleprep ID:	SP001944
Sampleprep Summary:	Each cell pellet was thawed on ice and resuspended in 500 μL ice-cold water by vortexing for 3 seconds and 500 μL of chilled (–80°C) 90% methanol + 10% chloroform solution was immediately added and vortexed for another 10 seconds and then kept on ice. Samples were incubated for 30 minutes at 4°C while rotating and then centrifuged at 800×g for 10 mins at 4°C. The supernatants were transferred to fresh tubes and centrifuged at 16000×g for 45 minutes at 4°C. The cleared supernatant containing metabolites were cleaned using a SPME (solid phase microextraction) protocol adopted from Mousavi et. al. (Mousavi et al., 2019), vacufuged to dryness and stored at –80°C. The cell pellets were used for protein extraction using GuHCl lysis method as described below.

Sampleprep ID:

SP001944

Sampleprep Summary:

Each cell pellet was thawed on ice and resuspended in 500 μL ice-cold water by vortexing for 3 seconds and 500 μL of chilled (–80°C) 90% methanol + 10% chloroform solution was immediately added and vortexed for another 10 seconds and then kept on ice. Samples were incubated for 30 minutes at 4°C while rotating and then centrifuged at 800×g for 10 mins at 4°C. The supernatants were transferred to fresh tubes and centrifuged at 16000×g for 45 minutes at 4°C. The cleared supernatant containing metabolites were cleaned using a SPME (solid phase microextraction) protocol adopted from Mousavi et. al. (Mousavi et al., 2019), vacufuged to dryness and stored at –80°C. The cell pellets were used for protein extraction using GuHCl lysis method as described below.

Combined analysis:

Analysis ID	AN003017
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Scientific EASY-nLC 1200 System
Column	Thermo Easy Spray
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	POSITIVE
Units	Neutral Mass

Chromatography:

Chromatography ID:	CH002235
Instrument Name:	Thermo Scientific EASY-nLC 1200 System
Column Name:	Thermo Easy Spray
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002806
Analysis ID:	AN003017
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	For metabolite identifications we used the R package MAIT (Fernández-Albert et al., 2014), which integrates peak detection, peak annotation and statistical analysis. Briefly, XCMS (Tautenhahn et al., 2012) is used to detect and align peaks followed by annotation with CAMERA (Kuhl et al., 2012). A special function ‘Biotransformations’ is applied to refine annotations and measured ions are then putatively identified by matching mass-to-charge ratios to a reference list of calculated masses of metabolites listed in the Human Metabolome Database (HMDB, http://www.hmdb.ca, 2019).
Ion Mode:	POSITIVE