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.

Study ID	ST002366
Study Title	Single cell lipidome analysis of phosphatidylcholines and spingomyelins from HepG2 and C2C12 cells
Study Type	Quantitative single cell lipidomics
Study Summary	We 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
Laboratory	Cellular Bioenergetics Laboratory
Last Name	Hancock
First Name	Sarah
Address	Lowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW, 2010, Australia
Email	s.hancock@victorchang.edu.au
Phone	+61414537526
Submit Date	2022-10-19
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	MS(Dir. Inf.)
Release Date	2023-10-19
Release Version	1

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
Species Group:	Mammals

Factors:

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

mb_sample_id	local_sample_id	Treatment	cell number	type
SA237061	HepG2-H08	CON	bulk_extract	sample
SA237062	HepG2-H09	CON	bulk_extract	sample
SA237063	HepG2-H07	CON	bulk_extract	sample
SA237064	C2C12-H07	CON	bulk_extract	sample
SA237065	C2C12-H09	CON	bulk_extract	sample
SA237066	C2C12-H08	CON	bulk_extract	sample
SA237067	HepG2-A01	CON	fifty_cells	sample
SA237068	C2C12-A02	CON	fifty_cells	sample
SA237069	C2C12-A03	CON	fifty_cells	sample
SA237070	C2C12-A01	CON	fifty_cells	sample
SA237071	HepG2-A03	CON	fifty_cells	sample
SA237072	HepG2-A02	CON	fifty_cells	sample
SA237073	HepG2-F02	CON	single_cells	sample
SA237074	HepG2-F03	CON	single_cells	sample
SA237075	C2C12-D01	CON	single_cells	sample
SA237076	HepG2-F01	CON	single_cells	sample
SA237077	C2C12-D02	CON	single_cells	sample
SA237078	C2C12-E01	CON	single_cells	sample
SA237079	C2C12-D03	CON	single_cells	sample
SA237080	C2C12-C03	CON	single_cells	sample
SA237081	HepG2-E02	CON	single_cells	sample
SA237082	HepG2-G01	CON	single_cells	sample
SA237083	C2C12-B03	CON	single_cells	sample
SA237084	C2C12-B02	CON	single_cells	sample
SA237085	C2C12-B01	CON	single_cells	sample
SA237086	C2C12-C01	CON	single_cells	sample
SA237087	HepG2-G03	CON	single_cells	sample
SA237088	HepG2-E01	CON	single_cells	sample
SA237089	HepG2-G02	CON	single_cells	sample
SA237090	C2C12-C02	CON	single_cells	sample
SA237091	HepG2-E03	CON	single_cells	sample
SA237092	C2C12-E02	CON	single_cells	sample
SA237093	HepG2-C02	CON	single_cells	sample
SA237094	HepG2-C03	CON	single_cells	sample
SA237095	C2C12-G02	CON	single_cells	sample
SA237096	C2C12-G03	CON	single_cells	sample
SA237097	HepG2-B01	CON	single_cells	sample
SA237098	HepG2-B02	CON	single_cells	sample
SA237099	HepG2-B03	CON	single_cells	sample
SA237100	C2C12-G01	CON	single_cells	sample
SA237101	HepG2-C01	CON	single_cells	sample
SA237102	C2C12-E03	CON	single_cells	sample
SA237103	C2C12-F03	CON	single_cells	sample
SA237104	C2C12-F01	CON	single_cells	sample
SA237105	HepG2-D03	CON	single_cells	sample
SA237106	C2C12-F02	CON	single_cells	sample
SA237107	HepG2-D01	CON	single_cells	sample
SA237108	HepG2-D02	CON	single_cells	sample
SA237109	HepG2-H10	DHA	bulk_extract	sample
SA237110	HepG2-H12	DHA	bulk_extract	sample
SA237111	C2C12-H10	DHA	bulk_extract	sample
SA237112	HepG2-H11	DHA	bulk_extract	sample
SA237113	C2C12-H11	DHA	bulk_extract	sample
SA237114	C2C12-H12	DHA	bulk_extract	sample
SA237115	HepG2-A08	DHA	fifty_cells	sample
SA237116	HepG2-A07	DHA	fifty_cells	sample
SA237117	C2C12-A09	DHA	fifty_cells	sample
SA237118	C2C12-A08	DHA	fifty_cells	sample
SA237119	C2C12-A07	DHA	fifty_cells	sample
SA237120	HepG2-A09	DHA	fifty_cells	sample
SA237121	C2C12-E09	DHA	single_cells	sample
SA237122	HepG2-D07	DHA	single_cells	sample
SA237123	HepG2-C09	DHA	single_cells	sample
SA237124	C2C12-E08	DHA	single_cells	sample
SA237125	HepG2-D08	DHA	single_cells	sample
SA237126	C2C12-D09	DHA	single_cells	sample
SA237127	HepG2-E07	DHA	single_cells	sample
SA237128	HepG2-D09	DHA	single_cells	sample
SA237129	HepG2-C08	DHA	single_cells	sample
SA237130	C2C12-E07	DHA	single_cells	sample
SA237131	C2C12-F08	DHA	single_cells	sample
SA237132	C2C12-G08	DHA	single_cells	sample
SA237133	C2C12-G09	DHA	single_cells	sample
SA237134	HepG2-B07	DHA	single_cells	sample
SA237135	C2C12-G07	DHA	single_cells	sample
SA237136	HepG2-B08	DHA	single_cells	sample
SA237137	C2C12-F09	DHA	single_cells	sample
SA237138	HepG2-C07	DHA	single_cells	sample
SA237139	HepG2-B09	DHA	single_cells	sample
SA237140	C2C12-F07	DHA	single_cells	sample
SA237141	C2C12-D08	DHA	single_cells	sample
SA237142	C2C12-B09	DHA	single_cells	sample
SA237143	HepG2-G07	DHA	single_cells	sample
SA237144	C2C12-B08	DHA	single_cells	sample
SA237145	C2C12-D07	DHA	single_cells	sample
SA237146	HepG2-G09	DHA	single_cells	sample
SA237147	HepG2-G08	DHA	single_cells	sample
SA237148	HepG2-F09	DHA	single_cells	sample
SA237149	C2C12-B07	DHA	single_cells	sample
SA237150	HepG2-E08	DHA	single_cells	sample
SA237151	HepG2-F08	DHA	single_cells	sample
SA237152	HepG2-F07	DHA	single_cells	sample
SA237153	HepG2-E09	DHA	single_cells	sample
SA237154	C2C12-C08	DHA	single_cells	sample
SA237155	C2C12-C07	DHA	single_cells	sample
SA237156	C2C12-C09	DHA	single_cells	sample
SA237157	HepG2-H06	N/A	bulk_extract	blank
SA237158	C2C12-H06	N/A	bulk_extract	blank
SA237159	C2C12-H02	N/A	single_cells	blank
SA237160	C2C12-H03	N/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.

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