Summary of Study ST003398

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 PR002104. The data can be accessed directly via it's Project DOI: 10.21228/M8NJ9Q 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.

Perform statistical analysis  |  Show all samples  |  Show named metabolites  |  Download named metabolite data  
Download mwTab file (text)   |  Download mwTab file(JSON)   |  Download data files (Contains raw data)
Study IDST003398
Study TitleSpecific activation of the integrated stress response (ISR) uncovers regulation of lipid droplet biogenesis
Study TypeBiology
Study SummaryU2OS cells were treated with Dimerizer-PERK for 0h,1h,2h,4h,8h and 24h. Lipidomics analysis using LC-MS was performed on these samples to understand the regulation of cellular lipidome upon ISR activation
Institute
Calico Life Sciences
DepartmentDepartment of Mass Spectrometry-Technology Lab
LaboratoryMetabolomics Lab
Last NameVu
First NameNgoc
Address1130 Veterans BLVD, South San Francisco, CA 94080
Emailngoc@calicolabs.com
Phone650-420-5430
Submit Date2024-08-08
Num Groups6
Total Subjects18
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2024-09-03
Release Version1
Ngoc Vu Ngoc Vu
https://dx.doi.org/10.21228/M8NJ9Q
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:


Project:

Project ID:PR002104
Project DOI:doi: 10.21228/M8NJ9Q
Project Title:Specific activation of the integrated stress response uncovers regulation of central carbon metabolism and lipid droplet biogenesis
Project Type:Biology
Project Summary:The integrated stress response (ISR) enables cells to cope with a variety of insults, but its specific contribution to downstream cellular outputs remains unclear. Using a synthetic tool, we selectively activate the ISR without co-activation of parallel pathways and define the resulting cellular state with multi-omics profiling. We identify time- and dose-dependent gene expression modules, with ATF4 driving only a small but sensitive subgroup that includes amino acid metabolic enzymes. This ATF4 response affects cellular bioenergetics, rerouting carbon utilization towards amino acid production and away from the tricarboxylic acid cycle and fatty acid synthesis. We also find an ATF4-independent reorganization of the lipidome that promotes DGAT-dependent triglyceride synthesis and accumulation of lipid droplets. While DGAT1 is the main driver of lipid droplet biogenesis, DGAT2 plays an essential role in buffering stress and maintaining cell survival. Together, we demonstrate the sufficiency of the ISR in promoting a previously unappreciated metabolic state.
Institute:Calico Life Sciences
Department:Department of Mass Spectrometry-Technology Lab
Laboratory:Metabolomics Lab
Last Name:Vu
First Name:Ngoc
Address:1130 Veterans BLVD, South San Francisco, CA 94080
Email:ngochmvu@gmail.com
Phone:6504205430
Publications:Labbe, Lebon & King et al., Specific activation of the integrated stress response uncovers regulation of central carbon metabolism and lipid droplet biogenesis.Nat Comm.2024

Subject:

Subject ID:SU003523
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Cell Strain Details:U2OS
Cell Counts:5e5

Factors:

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

mb_sample_id local_sample_id Time Sample source
SA3715130h_20 Human osteosarcoma U2-OS cells
SA3715140h_10 Human osteosarcoma U2-OS cells
SA3715150h_30 Human osteosarcoma U2-OS cells
SA3715161h_31 Human osteosarcoma U2-OS cells
SA3715171h_21 Human osteosarcoma U2-OS cells
SA3715181h_11 Human osteosarcoma U2-OS cells
SA37151924h_124 Human osteosarcoma U2-OS cells
SA37152024h_224 Human osteosarcoma U2-OS cells
SA37152124h_324 Human osteosarcoma U2-OS cells
SA3715222h_12 Human osteosarcoma U2-OS cells
SA3715232h_22 Human osteosarcoma U2-OS cells
SA3715242h_32 Human osteosarcoma U2-OS cells
SA3715254h_14 Human osteosarcoma U2-OS cells
SA3715264h_34 Human osteosarcoma U2-OS cells
SA3715274h_24 Human osteosarcoma U2-OS cells
SA3715288h_18 Human osteosarcoma U2-OS cells
SA3715298h_28 Human osteosarcoma U2-OS cells
SA3715308h_38 Human osteosarcoma U2-OS cells
SA371511DoubleBlank.mzML- Blank
SA371512std003.mzML- standard
Showing results 1 to 20 of 20

Collection:

Collection ID:CO003516
Collection Summary:The parental U2OS line was obtained from ATCC and engineered to express the Dmr-PERK construct in house.
Sample Type:human osteosarcoma cell line
Collection Method:Liquid Liquid Extraction- Matyash
Collection Location:Calico Life Sciences
Collection Frequency:1
Collection Duration:30 mins
Volumeoramount Collected:0.8 mL
Storage Conditions:-80℃
Collection Vials:2 mL glass vial
Storage Vials:2 mL glass vial
Collection Tube Temp:4C
Additives:none
Tissue Cell Identification:human osteosarcoma cell line

Treatment:

Treatment ID:TR003532
Treatment Summary:U2OS cells were treated with 0.05nM of dimerizer over the course of 0,1,2,4,8,24 hours for lipidomics analysis
Treatment Compound:DImerizer AP20187
Treatment Dose:0.05 nM to media
Treatment Doseduration:0h,1h,2h,4h,8h,24h
Treatment Vehicle:ethanol
Cell Growth Container:6-well plate
Cell Growth Config:U2OS cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1X antibiotic-antimycotic solution (Gibco). Cells were housed in an incubator at 37°C, 5% CO2, 20% O2 and passaged every 2-3 days with trypsin.
Cell Media:U2OS cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1X antibiotic-antimycotic solution (Gibco).
Cell Harvesting:LLE-MTBE (Matyash)
Cell Pct Confluence:80%
Cell Media Lastchanged:24 hours prior to harvesting

Sample Preparation:

Sampleprep ID:SP003530
Sampleprep Summary:Cells were cultured in 6-well plates, and ~500,000 cells per well were washed with warm PBS and quenched with 0.8 mL of 50/50 MeOH/H2O containing 100 µL of SPLASH LIPIDOMIXLipidSplash internal standards (Avanti Polar Lipids), followed by incubation at -20℃ for 15 minutes. Samples were scraped and collected to a 2 mL glass vial and extracted for lipidomics analysis using an MTBE-LLE extraction adopted from Matyash, et al 87. In brief, 800 μL of MTBE was added to the mixture, vortexed for 30 seconds and incubated on ice for another 15 minutes and centrifuged at 3500 RPM for 10 minutes at 4℃. Lipids partitioned in the top layer were collected into a separate vial, and the extraction process was repeated with an addition of 600 µL of MTBE. After incubating and centrifuging, the second organic layer was collected and combined in the first lipid vial, dried under nitrogen at 4℃, resuspended in 200 µL of ButOH/MeOH/H2O (2:1:1, v/v/v) for lipid analysis.
Processing Storage Conditions:-80℃
Extraction Method:Matyash
Extract Storage:4℃
Sample Resuspension:ButOH/MeOH/H2O (2:1:1, v/v/v)

Combined analysis:

Analysis ID AN005577 AN005578
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish
Column Thermo Scientific Accucore C30 (250 × 2.1mm, 2.6um, 150 Å) Thermo Scientific Accucore C30 (250 × 2.1mm, 2.6um, 150 Å)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Plus Orbitrap Thermo Q Exactive Plus Orbitrap
Ion Mode POSITIVE NEGATIVE
Units Log2(Top Peak Area) Log2(Top Peak Area)

Chromatography:

Chromatography ID:CH004238
Chromatography Summary:RPLC with a Thermo Accucore C30 for lipidomics analysis
Instrument Name:Thermo Vanquish
Column Name:Thermo Scientific Accucore C30 (250 × 2.1mm, 2.6um, 150 Å)
Column Pressure:900 bar
Column Temperature:35
Flow Gradient:The gradient was t(min) = −7, 30% B, t = 0, 30% B, t = 7, 43% B, t = 12, 65% B, t = 30, 70% B, t = 31, 88% B, t = 51, 95% B, t = 53, 100% B, t = 55, 100% B, t = 55.1, 30% B, t = 60, 30% B.
Flow Rate:200uL/min
Injection Temperature:4C
Internal Standard:Splash Lipidomix (Avanti Polar Lipids)
Solvent A:60% acetonitrile/40% water; 20mM ammonium formate; 0.25μM medronic acid
Solvent B:90% isopropanol/10% acetonitrile; 20mM ammonium formate; 0.25μM medronic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS005302
Analysis ID:AN005577
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The mass spectrometer settings were as follows: data-dependent acquisition (DDA) was performed with the following parameters: resolution = 140,000, AGC target = 3.00 × 106, maximum IT (ms) = 100, scan range = 200–2000. The MS2 parameters were as follows: resolution = 17,500, AGC target = 3.00 × 106, maximum IT (ms) = 150, loop count = 8, isolation window (m/z) = 1, (N)CE = 20, 30, 40; underfill ratio = 1.00%, Apex trigger(s) = 5–30, dynamic exclusion(s) = 15 s. Maven software was used for data processing (Seitzer, P., Bennett, B. & Melamud, E. MAVEN2: An Updated Open-Source Mass Spectrometry Exploration Platform. Metabolites 12, 684 (2022).) Analysis of the lipidomics and metabolomics data was performed using Calico Lipidomics and Metabolomics Analysis (clamanR) package (https://github.com/calico/claman) in R. Raw files were converted to the mxML format using ProteoWizard Ver 3 88. Compound identifications were detected and grouped using the OpenCLaM R package (https://github.com/calico/open_clam) and manually inspected using the MAVEN2 peak analysis program (https://github.com/eugenemel/maven) 85 with the criteria of a precursor ion tolerance of 10 ppm and a product ion tolerance of 20 ppm, comparing fragmentation and retention time to an in-house library generated from authentic standards for metabolomics, and in-house generated in-silico libraries for lipidomics (https://github.com/calico/CalicoLipidLibrary). Per feature, our software assigned a unique groupID in a numerical format.
Ion Mode:POSITIVE
  
MS ID:MS005303
Analysis ID:AN005578
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The mass spectrometer settings were as follows: data-dependent acquisition (DDA) was performed with the following parameters: resolution = 140,000, AGC target = 3.00 × 106, maximum IT (ms) = 100, scan range = 200–2000. The MS2 parameters were as follows: resolution = 17,500, AGC target = 3.00 × 106, maximum IT (ms) = 150, loop count = 8, isolation window (m/z) = 1, (N)CE = 20, 30, 40; underfill ratio = 1.00%, Apex trigger(s) = 5–30, dynamic exclusion(s) = 15 s. Maven software was used for data processing (Seitzer, P., Bennett, B. & Melamud, E. MAVEN2: An Updated Open-Source Mass Spectrometry Exploration Platform. Metabolites 12, 684 (2022).) Analysis of the lipidomics and metabolomics data was performed using Calico Lipidomics and Metabolomics Analysis (clamanR) package (https://github.com/calico/claman) in R. Raw files were converted to the mxML format using ProteoWizard Ver 3 88. Compound identifications were detected and grouped using the OpenCLaM R package (https://github.com/calico/open_clam) and manually inspected using the MAVEN2 peak analysis program (https://github.com/eugenemel/maven) 85 with the criteria of a precursor ion tolerance of 10 ppm and a product ion tolerance of 20 ppm, comparing fragmentation and retention time to an in-house library generated from authentic standards for metabolomics, and in-house generated in-silico libraries for lipidomics (https://github.com/calico/CalicoLipidLibrary). Per feature, our software assigned a unique groupID in a numerical format.
Ion Mode:NEGATIVE
  logo