Summary of Study ST002366

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 PR001521. The data can be accessed directly via it's Project DOI: 10.21228/M83707 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 IDST002366
Study TitleSingle cell lipidome analysis of phosphatidylcholines and spingomyelins from HepG2 and C2C12 cells
Study TypeQuantitative single cell lipidomics
Study SummaryWe have developed a high-throughput method for the detection and quantification of a wide range of phosphatidylcholine (PC) and sphingomyelin (SM) species from single cells that combines fluorescence-assisted cell sorting (FACS) with automated chip-based nanoelectrospray ionization (nanoESI) and shotgun lipidomics. Using this method we can detect and perform relative quantitation on more than >50 different PC and SM species from immortalised human cells, and can easily distinguish between cells of different lineages (e.g. hepatocarcinoma HePG2 vs C2C12 myoblasts) and cells treated with exogenous fatty acids.
Institute
Victor Chang Cardiac Research Institute
LaboratoryCellular Bioenergetics Laboratory
Last NameHancock
First NameSarah
AddressLowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW, 2010, Australia
Emails.hancock@victorchang.edu.au
Phone+61414537526
Submit Date2022-10-19
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailMS(Dir. Inf.)
Release Date2023-10-19
Release Version1
Sarah Hancock Sarah Hancock
https://dx.doi.org/10.21228/M83707
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001521
Project DOI:doi: 10.21228/M83707
Project Title:Single cell lipidomics
Project Type:Quantitative lipidomics
Project Summary:Analysis of lipids from single cells isolated by FACS.
Institute:Victor Chang Cardiac Research Institute
Laboratory:Cellular Bioenergetics Laboratory
Last Name:Hancock
First Name:Sarah
Address:Lowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW, Australia 2010
Email:s.hancock@victorchang.edu.au
Phone:+61414537526
Funding Source:PdCCRS
Project Comments:Study 1 of 2

Subject:

Subject ID:SU002455
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

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

mb_sample_id local_sample_id Treatment cell number type
SA237061HepG2-H08CON bulk_extract sample
SA237062HepG2-H09CON bulk_extract sample
SA237063HepG2-H07CON bulk_extract sample
SA237064C2C12-H07CON bulk_extract sample
SA237065C2C12-H09CON bulk_extract sample
SA237066C2C12-H08CON bulk_extract sample
SA237067HepG2-A01CON fifty_cells sample
SA237068C2C12-A02CON fifty_cells sample
SA237069C2C12-A03CON fifty_cells sample
SA237070C2C12-A01CON fifty_cells sample
SA237071HepG2-A03CON fifty_cells sample
SA237072HepG2-A02CON fifty_cells sample
SA237073HepG2-F02CON single_cells sample
SA237074HepG2-F03CON single_cells sample
SA237075C2C12-D01CON single_cells sample
SA237076HepG2-F01CON single_cells sample
SA237077C2C12-D02CON single_cells sample
SA237078C2C12-E01CON single_cells sample
SA237079C2C12-D03CON single_cells sample
SA237080C2C12-C03CON single_cells sample
SA237081HepG2-E02CON single_cells sample
SA237082HepG2-G01CON single_cells sample
SA237083C2C12-B03CON single_cells sample
SA237084C2C12-B02CON single_cells sample
SA237085C2C12-B01CON single_cells sample
SA237086C2C12-C01CON single_cells sample
SA237087HepG2-G03CON single_cells sample
SA237088HepG2-E01CON single_cells sample
SA237089HepG2-G02CON single_cells sample
SA237090C2C12-C02CON single_cells sample
SA237091HepG2-E03CON single_cells sample
SA237092C2C12-E02CON single_cells sample
SA237093HepG2-C02CON single_cells sample
SA237094HepG2-C03CON single_cells sample
SA237095C2C12-G02CON single_cells sample
SA237096C2C12-G03CON single_cells sample
SA237097HepG2-B01CON single_cells sample
SA237098HepG2-B02CON single_cells sample
SA237099HepG2-B03CON single_cells sample
SA237100C2C12-G01CON single_cells sample
SA237101HepG2-C01CON single_cells sample
SA237102C2C12-E03CON single_cells sample
SA237103C2C12-F03CON single_cells sample
SA237104C2C12-F01CON single_cells sample
SA237105HepG2-D03CON single_cells sample
SA237106C2C12-F02CON single_cells sample
SA237107HepG2-D01CON single_cells sample
SA237108HepG2-D02CON single_cells sample
SA237109HepG2-H10DHA bulk_extract sample
SA237110HepG2-H12DHA bulk_extract sample
SA237111C2C12-H10DHA bulk_extract sample
SA237112HepG2-H11DHA bulk_extract sample
SA237113C2C12-H11DHA bulk_extract sample
SA237114C2C12-H12DHA bulk_extract sample
SA237115HepG2-A08DHA fifty_cells sample
SA237116HepG2-A07DHA fifty_cells sample
SA237117C2C12-A09DHA fifty_cells sample
SA237118C2C12-A08DHA fifty_cells sample
SA237119C2C12-A07DHA fifty_cells sample
SA237120HepG2-A09DHA fifty_cells sample
SA237121C2C12-E09DHA single_cells sample
SA237122HepG2-D07DHA single_cells sample
SA237123HepG2-C09DHA single_cells sample
SA237124C2C12-E08DHA single_cells sample
SA237125HepG2-D08DHA single_cells sample
SA237126C2C12-D09DHA single_cells sample
SA237127HepG2-E07DHA single_cells sample
SA237128HepG2-D09DHA single_cells sample
SA237129HepG2-C08DHA single_cells sample
SA237130C2C12-E07DHA single_cells sample
SA237131C2C12-F08DHA single_cells sample
SA237132C2C12-G08DHA single_cells sample
SA237133C2C12-G09DHA single_cells sample
SA237134HepG2-B07DHA single_cells sample
SA237135C2C12-G07DHA single_cells sample
SA237136HepG2-B08DHA single_cells sample
SA237137C2C12-F09DHA single_cells sample
SA237138HepG2-C07DHA single_cells sample
SA237139HepG2-B09DHA single_cells sample
SA237140C2C12-F07DHA single_cells sample
SA237141C2C12-D08DHA single_cells sample
SA237142C2C12-B09DHA single_cells sample
SA237143HepG2-G07DHA single_cells sample
SA237144C2C12-B08DHA single_cells sample
SA237145C2C12-D07DHA single_cells sample
SA237146HepG2-G09DHA single_cells sample
SA237147HepG2-G08DHA single_cells sample
SA237148HepG2-F09DHA single_cells sample
SA237149C2C12-B07DHA single_cells sample
SA237150HepG2-E08DHA single_cells sample
SA237151HepG2-F08DHA single_cells sample
SA237152HepG2-F07DHA single_cells sample
SA237153HepG2-E09DHA single_cells sample
SA237154C2C12-C08DHA single_cells sample
SA237155C2C12-C07DHA single_cells sample
SA237156C2C12-C09DHA single_cells sample
SA237157HepG2-H06N/A bulk_extract blank
SA237158C2C12-H06N/A bulk_extract blank
SA237159C2C12-H02N/A single_cells blank
SA237160C2C12-H03N/A single_cells blank
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Collection:

Collection ID:CO002448
Collection Summary:Samples were obtained from established immortalised adherent human cell lines HepG2 (hepatocarcinoma) and C2C12 (myoblasts). Cells were cultured in high glucose DMEM (D6429, Sigma-Aldrich) with 10% heat-inactivated fetal calf serum and incubated at 37°C in 5% CO2. Cell media was changed every three days and cells were passaged regularly at ~80-90% confluency by trypsinization. Cell lines were regularly screened for mycoplasma infection.
Sample Type:Adherent cell lines

Treatment:

Treatment ID:TR002467
Treatment Summary:Cells were treated by applying docosahexaenoic acid (DHA) conjugated to bovine serum albumin (BSA). DHA was obtained from Avanti Polar lipids (Alabaster, AL, USA) and dissolved in 100% ethanol at a concentration of 100 mM. 25 μL of DHA stock or 25 μl of 100% ethanol (CON) was added per 50 mL of normal growth media containing 2% (w/v) of fatty acid-free BSA. Media was sterile filtered by passing it through a 0.2 μM PES filter, and then both CON and DHA-containing media were incubated in a water bath for 2 hours at 55ºC to conjugate DHA to BSA. Conjugated media was stored at 4ºC until used. Cells were incubated overnight in the presence of pre-warmed CON or DHA-conjugated media before harvest.

Sample Preparation:

Sampleprep ID:SP002461
Sampleprep Summary:Samples were prepared one of three ways: as bulk extracts from ~1 million cells, or from fluorescence-assisted cell sorting (FACS) into either pooled fifty cells samples or singly-isolated cells. Full sample prep details are provided in the attached protocol.
Sampleprep Protocol Filename:MWB_sampleprep_protocol.pdf

Combined analysis:

Analysis ID AN003861
Analysis type MS
Chromatography type None (Direct infusion)
Chromatography system none
Column none
MS Type ESI
MS instrument type Triple quadrupole
MS instrument name ABI Sciex 5500 QTrap
Ion Mode POSITIVE
Units Peak Area (cps)

Chromatography:

Chromatography ID:CH002859
Instrument Name:none
Column Name:none
Chromatography Type:None (Direct infusion)

MS:

MS ID:MS003602
Analysis ID:AN003861
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:NanoESI mass spectrometry of lipid extracts was performed using a hybrid triple quadrupole linear ion trap mass spectrometer (QTRAP® 5500, SCIEX, Framingham, MA, USA) equipped with an automated chip-based nanoelectrospray source (TriVersa Nanomate®, Advion Biosciences, NY, USA). Spray parameters were set at a gas pressure of 0.4 psi and a voltage of 1.2 kV. PC and SM data were acquired in positive ion mode using a precursor ion scan of m/z 184 at a scan rate of 200 Da/s across a mass range of 640 – 850 m/z. Declustering potential was set at 100 V, entrance potential at 10 V, collision energy at 47 V, and collision cell exit potential at 8V (31). Aspiration of 10 μl of sample from each well generated a stable spray time of ≥30 minutes. Lipids were identified from acquired data using Lipidview™ software (v1.2b, SCIEX, Framingham, MA, USA). Processing settings in Lipidview™ were set at a mass tolerance of 0.5 Da, with a minimum intensity of 0.1% and a minimum signal-to-noise ratio of 4. Smoothing and deisotoping of lipid species were enabled. Lipid species were identified from target lists (see attached protocol files), and peak area for each detected lipid species was then exported. These data underwent further processing in R, including background subtraction and relative quantification from internal standards. Lipid nomenclature follows recommendations for the level of molecular detail known (as per Liebisch et al. 2013 J Lipid Res); and in the present study we report lipids as class (e.g., PC or SM) followed by the total number of carbons and carbon-carbon double bonds present within the fatty acids separated by a colon (e.g., a PC with 34 carbons and 1 double bond as PC 34:1). At the level of identification available by the technique used in this study some ambiguity exists between isobaric PC species containing either odd-chain or ether-linked fatty acid species, and in the absence of further structural detail we chose to report such species as ether-linked only where overlap exists.
Ion Mode:POSITIVE
Analysis Protocol File:MWB_Lipidview_target_list.txt
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