Summary of Study ST001861

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001174. The data can be accessed directly via it's Project DOI: 10.21228/M8Z98Q This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study ID	ST001861
Study Title	Parallelized multidimensional analytic framework, PAMAF, applied to mammalian cells uncovers novel regulatory principles in EMT
Study Summary	Painting a holistic picture of disease etiology will require longitudinal systems-scale reconstruction of the multitiered architecture of eukaryotic signaling. As opposed to ‘one omic at a time’, which provides an incomplete view on disease mechanisms, here we developed an experimental and analytics framework, PAMAF, to simultaneously acquire and analyze twelve omic modalities from the same set of samples, i.e., protein abundance from whole-cells, nucleus, exosomes, secretome and membrane; peptidome; N-glycosylation, phosphorylation; metabolites; mRNA, miRNA; and, in parallel, single-cell transcriptomes. We applied PAMAF in a well-studied in vitro model of TGFβ-induced EMT to generate the EMT-ExMap dataset, cataloguing >61,000 expression profiles (>10,000 differential) over 12 days. PAMAF revealed that EMT is more complex than currently understood and identified numerous stage-specific mechanisms and vulnerabilities not captured in literature. Broad application of PAMAF will provide unprecedented insights into multifaceted biological processes relevant to human health and disease.
Institute	Boston University
Last Name	Paul
First Name	Indranil
Address	71 East Concord St
Email	indranil@bu.edu
Phone	6177929632
Submit Date	2021-06-22
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2022-11-11
Release Version	1

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Project:

Project ID:	PR001174
Project DOI:	doi: 10.21228/M8Z98Q
Project Title:	A multi-tiered map of EMT defines major transition points and identifies vulnerabilities
Project Summary:	Epithelial to mesenchymal transition (EMT) is a complex cellular program proceeding through a hybrid E/M state linked to cancer-associated stemness, migration and chemoresistance. Deeper molecular understanding of this dynamic physiological landscape is needed to define events which regulate the transition and entry into and exit from the E/M state. Here, we quantified >60,000 molecules across ten time points and twelve omic layers in human mammary epithelial cells undergoing TGFβ-induced EMT. Deep proteomic profiles of whole cells, nuclei, extracellular vesicles, secretome, membrane and phosphoproteome defined state-specific signatures and major transition points. Parallel metabolomics showed metabolic reprogramming preceded changes in other layers, while single-cell RNA sequencing identified transcription factors controlling entry into E/M. Covariance analysis exposed unexpected discordance between the molecular layers. Integrative causal modeling revealed co-dependencies governing entry into E/M that were verified experimentally using combinatorial inhibition. Overall, this dataset provides an unprecedented resource on TGFβ signaling, EMT and cancer.
Institute:	Boston University
Last Name:	Paul
First Name:	Indranil
Address:	71 East Concord Street, Room # K320
Email:	indranil@bu.edu
Phone:	6177929631

Subject:

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

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Replicate	Treatment	Treatment
SA174345	20190525_Indranil_pos_spme_meta1_1	1	Control	0_day
SA174346	20190525_Indranil_pos_spme_meta10_1	1	TGFbeta	12_day
SA174347	20190525_Indranil_pos_spme_meta3_1	1	TGFbeta	1_day
SA174348	20190525_Indranil_pos_spme_meta4_1	1	TGFbeta	2_day
SA174349	20190525_Indranil_pos_spme_meta5_1	1	TGFbeta	3_day
SA174350	20190525_Indranil_pos_spme_meta6_1	1	TGFbeta	4_day
SA174351	20190525_Indranil_pos_spme_meta2_1	1	TGFbeta	4_hours
SA174352	20190525_Indranil_pos_spme_meta7_1	1	TGFbeta	5_day
SA174353	20190525_Indranil_pos_spme_meta8_1	1	TGFbeta	6_day
SA174354	20190525_Indranil_pos_spme_meta9_1	1	TGFbeta	8_day
SA174355	20190525_Indranil_pos_spme_meta1_2	2	Control	0_day
SA174356	20190525_Indranil_pos_spme_meta10_2	2	TGFbeta	12_day
SA174357	20190525_Indranil_pos_spme_meta3_2	2	TGFbeta	1_day
SA174358	20190525_Indranil_pos_spme_meta4_2	2	TGFbeta	2_day
SA174359	20190525_Indranil_pos_spme_meta5_2	2	TGFbeta	3_day
SA174360	20190525_Indranil_pos_spme_meta6_2	2	TGFbeta	4_day
SA174361	20190525_Indranil_pos_spme_meta2_2	2	TGFbeta	4_hours
SA174362	20190525_Indranil_pos_spme_meta7_2	2	TGFbeta	5_day
SA174363	20190525_Indranil_pos_spme_meta8_2	2	TGFbeta	6_day
SA174364	20190525_Indranil_pos_spme_meta9_2	2	TGFbeta	8_day
SA174365	20190525_Indranil_pos_spme_meta1_3	3	Control	0_day
SA174366	20190525_Indranil_pos_spme_meta10_3	3	TGFbeta	12_day
SA174367	20190525_Indranil_pos_spme_meta3_3	3	TGFbeta	1_day
SA174368	20190525_Indranil_pos_spme_meta4_3	3	TGFbeta	2_day
SA174369	20190525_Indranil_pos_spme_meta5_3	3	TGFbeta	3_day
SA174370	20190525_Indranil_pos_spme_meta6_3	3	TGFbeta	4_day
SA174371	20190525_Indranil_pos_spme_meta2_3	3	TGFbeta	4_hours
SA174372	20190525_Indranil_pos_spme_meta7_3	3	TGFbeta	5_day
SA174373	20190525_Indranil_pos_spme_meta8_3	3	TGFbeta	6_day
SA174374	20190525_Indranil_pos_spme_meta9_3	3	TGFbeta	8_day

Showing results 1 to 30 of 30

Showing results 1 to 30 of 30

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