Summary of Study ST003758

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 PR002341. The data can be accessed directly via it's Project DOI: 10.21228/M81N8S This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST003758
Study Title	Untargeted lipidomics of gemcitabine-resistant cells
Study Summary	In this work we developed gemcitabine (GEMC) resistant (GEMR) PDAC cell lines from Panc1 and MiaPaCa2 cells. This dataset contains results and raw data for untargeted lipidomics of Panc1 cells, including control (CON), and GEMR cells alongside internal quality controls (iQC) and blank samples for background subtraction (as described under sample prep). Untargeted lipidomics enabled the quantification of more than 400 distinct molecular lipid species within Panc1 & MiaPaCa2 control (CON) & GEMR cells from 12 different classes of lipid, with 79 molecular species being differentially abundant between MiaPaCa2 control and GEMR cells following unpaired t test. Data were analysed using MS-DIAL 4.9.
Institute	Victor Chang Cardiac Research Institute
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	2025-02-19
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2025-03-21
Release Version	1

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Project:

Project ID:	PR002341
Project DOI:	doi: 10.21228/M81N8S
Project Title:	Untargeted lipidomics of gemcitabine-resistant PDAC cells
Project Summary:	Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with few treatment options and poor survivability. In this work we sought to characterise metabolic adaptations to gemcitabine (GEMC)-based chemotherapy exposure to discover new therapeutic targets for improving treatment efficacy. We show that GEMC resistance (GEMR) upregulates de novo lipogenesis in Panc1 and MiaPaCa2 cells.
Institute:	Victor Chang Cardiac Research Institute
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

Subject:

Subject ID:	SU003891
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	Sample source	group	group
SA409065	Blank_cells_neg_20211210204022	-	Blank	Blank
SA409066	Blank_cells_neg	-	Blank	Blank
SA409067	Blank_cells_pos_20211210152840	-	Blank	Blank
SA409068	Blank_cells_pos	-	Blank	Blank
SA409069	iQC_cells_neg_20211210211133	-	pool	iQC
SA409070	iQC_cells_pos	-	pool	iQC
SA409071	iQC_cells_pos_20211210155952	-	pool	iQC
SA409072	iQC_cells_neg	-	pool	iQC
SA409073	MiaPaCa2_CON_3_pos	Pancreas	MiaPaCa2 cells	CON
SA409074	MiaPaCa2_CON_3_neg	Pancreas	MiaPaCa2 cells	CON
SA409075	MiaPaCa2_CON_2_pos	Pancreas	MiaPaCa2 cells	CON
SA409076	MiaPaCa2_CON_1_pos	Pancreas	MiaPaCa2 cells	CON
SA409077	MiaPaCa2_CON_1_neg	Pancreas	MiaPaCa2 cells	CON
SA409078	MiaPaCa2_CON_2_neg	Pancreas	MiaPaCa2 cells	CON
SA409079	MiaPaCa2_GEMR_1_neg	Pancreas	MiaPaCa2 cells	GEMR
SA409080	MiaPaCa2_GEMR_1_pos	Pancreas	MiaPaCa2 cells	GEMR
SA409081	MiaPaCa2_GEMR_2_neg	Pancreas	MiaPaCa2 cells	GEMR
SA409082	MiaPaCa2_GEMR_2_pos	Pancreas	MiaPaCa2 cells	GEMR
SA409083	MiaPaCa2_GEMR_3_neg	Pancreas	MiaPaCa2 cells	GEMR
SA409084	MiaPaCa2_GEMR_3_pos	Pancreas	MiaPaCa2 cells	GEMR

Showing results 1 to 20 of 20

Showing results 1 to 20 of 20

Collection:

Collection ID:	CO003884
Collection Summary:	Samples were obtained from established immortalised adherent human pancreatic ductal adenocarcinoma cells (MiaPaCa2). Cells were cultured in high glucose DMEM containing 4.5 g/L glucose and 4 mM glutamine and were supplemented with 10% FCS. Cells were cultured under 5% CO2 at 37ºC and routinely passaged every 2-3 days (when ~80-90% confluent). Cell lines were regularly screened for mycoplasma infection.
Sample Type:	Pancreas

Treatment:

Treatment ID:	TR003900
Treatment Summary:	MiaPaCa2 GEMR cells were produced by serial treatment with escalating doses of gemcitabine for approximately 12 weeks. The final concentration of GEMC for MiaPaCa2 cells was 64 nM, resulting in a half-maximal inhibitory concentration of 92.2 nM (approximately 3x increase over control (CON) cells, 30.6 nM)

Sample Preparation:

Sampleprep ID:	SP003897
Sampleprep Summary:	Lipids were extracted from cells grown to 80-90% confluency in 6 well plates using a modified methyl tert-butyl ether (MTBE) method. Adherent cells were washed with ice-cold PBS and scraped into methanol containing 0.01% butylated hydroxytoluene and 100 pmol each of internal standards (phosphatidylcholine 19:0/19:0, phosphatidylethanolamine 17:0/17:0, phosphatidylserine 17:0/17:0, lysophosphatidylcholine 17:0, lysophosphatidylethanolamine 14:0, phosphatidylglycerol 17:0/17:0, phosphatidic acid 17:0/17:0, ceramide d18:1/17:0, dihydrosphingomyelin 12:0, cholesteryl ester 22:1, diacylglycerol 17:0/17:0, D5-triacylglycerol 16:0/16:0/16:0 and cardiolipin 14:0/14:0/14:0/14:0). An empty well was also scraped for use in background subtraction. To this, MTBE was added at a ratio of 3:1 v/v, and extracts were rotated overnight at 4°C. Following overnight rotation, 1 part of ice-cold 150 mM ammonium acetate was added, samples were vortexed vigorously and centrifuged at 2000 x g for 5 min. The upper organic phase containing lipids was transferred into a 1.5 mL autosampler vial and dried under N2 at 37ºC Dried lipids were reconstituted in chloroform:methanol:water (60:30:4.5 v/v/v), transferred to a sleeved vial, and stored at -20°C until analysis. The aqueous phase containing the protein pellet was dried under N2 and digested with 1M NaOH overnight at 4ºC. Digested protein was diluted 1:2 v/v with water and protein concentration was determined using a Pierce BCA assay kit (ThermoFisher Scientific)

Sampleprep ID:

SP003897

Sampleprep Summary:

Lipids were extracted from cells grown to 80-90% confluency in 6 well plates using a modified methyl tert-butyl ether (MTBE) method. Adherent cells were washed with ice-cold PBS and scraped into methanol containing 0.01% butylated hydroxytoluene and 100 pmol each of internal standards (phosphatidylcholine 19:0/19:0, phosphatidylethanolamine 17:0/17:0, phosphatidylserine 17:0/17:0, lysophosphatidylcholine 17:0, lysophosphatidylethanolamine 14:0, phosphatidylglycerol 17:0/17:0, phosphatidic acid 17:0/17:0, ceramide d18:1/17:0, dihydrosphingomyelin 12:0, cholesteryl ester 22:1, diacylglycerol 17:0/17:0, D5-triacylglycerol 16:0/16:0/16:0 and cardiolipin 14:0/14:0/14:0/14:0). An empty well was also scraped for use in background subtraction. To this, MTBE was added at a ratio of 3:1 v/v, and extracts were rotated overnight at 4°C. Following overnight rotation, 1 part of ice-cold 150 mM ammonium acetate was added, samples were vortexed vigorously and centrifuged at 2000 x g for 5 min. The upper organic phase containing lipids was transferred into a 1.5 mL autosampler vial and dried under N2 at 37ºC Dried lipids were reconstituted in chloroform:methanol:water (60:30:4.5 v/v/v), transferred to a sleeved vial, and stored at -20°C until analysis. The aqueous phase containing the protein pellet was dried under N2 and digested with 1M NaOH overnight at 4ºC. Digested protein was diluted 1:2 v/v with water and protein concentration was determined using a Pierce BCA assay kit (ThermoFisher Scientific)

Combined analysis:

Analysis ID	AN006170	AN006171
Chromatography ID	CH004685	CH004685
MS ID	MS005875	MS005876
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000	Thermo Dionex Ultimate 3000
Column	Waters ACQUITY UPLC CSH C18 (100 x 2.1 mm, 1.7 μm)	Waters ACQUITY UPLC CSH C18 (100 x 2.1 mm, 1.7 μm)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	nmol/mg protein	nmol/mg protein

Chromatography:

Chromatography ID:	CH004685
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Waters ACQUITY UPLC CSH C18 (100 x 2.1 mm, 1.7 μm)
Column Temperature:	60°C
Flow Gradient:	0.1-1.5 min 68% A (32% B), 1.4-4 min 55% A (45% B), 4-5 min 48% A (52% B), 5-8 min 42% A (58% B), 8-11 min 34% A (66% B), 11-14 min 30% A (70% B), 14-18 min 25% A (75% B), 18-21 min 3% A (97% B), 21-25 min 3% A (97% B), 25.1-30 min 68% A (32% B).
Flow Rate:	0.26 mL/min
Solvent A:	60% Acetonitrile/40% Water; 10 mM Ammonium Formate; 0.1% Formic acid
Solvent B:	10% Acetonitrile/90% Isopropanol; 10 mM Ammonium Formate; 0.1% Formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005875
Analysis ID:	AN006170
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Source conditions were as follows: a spray voltage 4.0 kV in positive ion mode and 3.5 kV in negative ion mode, capillary temperature 290°C, S lens RF 50, and auxiliary gas heater temperature of 250°C. Nitrogen was used as both source and collision gas, with the sheath gas flow rate set at 20 and the auxiliary gas flow rate at 5 (arbitrary units). Data were acquired in full scan/data-dependent MS2 mode (full-scan resolution 70,000 FWHM, max ion injection time 50 ms, scan range m/z 200–1,500), with the 10 most abundant ions being subjected to collision-induced dissociation using an isolation window of 1.5 Da and a normalized stepped collision energy of 15/27 eV. Product ions were detected at a resolution of 17,500. An exclusion list for background ions was developed using extraction blanks, and mass calibration was performed in both positive and negative ionization modes before analysis to ensure mass accuracy of 5 ppm in full-scan mode.
Ion Mode:	POSITIVE

MS ID:	MS005876
Analysis ID:	AN006171
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Source conditions were as follows: a spray voltage 4.0 kV in positive ion mode and 3.5 kV in negative ion mode, capillary temperature 290°C, S lens RF 50, and auxiliary gas heater temperature of 250°C. Nitrogen was used as both source and collision gas, with the sheath gas flow rate set at 20 and the auxiliary gas flow rate at 5 (arbitrary units). Data were acquired in full scan/data-dependent MS2 mode (full-scan resolution 70,000 FWHM, max ion injection time 50 ms, scan range m/z 200–1,500), with the 10 most abundant ions being subjected to collision-induced dissociation using an isolation window of 1.5 Da and a normalized stepped collision energy of 15/27 eV. Product ions were detected at a resolution of 17,500. An exclusion list for background ions was developed using extraction blanks, and mass calibration was performed in both positive and negative ionization modes before analysis to ensure mass accuracy of 5 ppm in full-scan mode.
Ion Mode:	NEGATIVE