#METABOLOMICS WORKBENCH shancock_20250221_204759 DATATRACK_ID:5666 STUDY_ID:ST003764 ANALYSIS_ID:AN006179 PROJECT_ID:PR002347
VERSION             	1
CREATED_ON             	February 21, 2025, 9:24 pm
#PROJECT
PR:PROJECT_TITLE                 	Profiling of fatty acid double bond positing ing gemcitabine-resistant
PR:PROJECT_TITLE                 	pancreatic ductal adenocarcinoma cells
PR:PROJECT_SUMMARY               	Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with few
PR:PROJECT_SUMMARY               	treatment options and poor survivability. In this work we sought to characterise
PR:PROJECT_SUMMARY               	metabolic adaptations to gemcitabine (GEMC)-based chemotherapy exposure to
PR:PROJECT_SUMMARY               	discover new therapeutic targets for improving treatment efficacy. We show that
PR:PROJECT_SUMMARY               	GEMC resistance (GEMR) upregulates de novo lipogenesis in Panc1 and MiaPaCa2
PR:PROJECT_SUMMARY               	cells.
PR:INSTITUTE                     	Victor Chang Cardiac Research Institute
PR:LAST_NAME                     	Hancock
PR:FIRST_NAME                    	Sarah
PR:ADDRESS                       	Level 7 Lowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW 2010,
PR:ADDRESS                       	Australia
PR:EMAIL                         	s.hancock@victorchang.edu.au
PR:PHONE                         	+61414537526
#STUDY
ST:STUDY_TITLE                   	Profiling of fatty acid double bond positing ing gemcitabine-resistant
ST:STUDY_TITLE                   	pancreatic ductal adenocarcinoma cells
ST:STUDY_SUMMARY                 	In this work we developed gemcitabine (GEMC) resistant (GEMR) PDAC cell lines
ST:STUDY_SUMMARY                 	from Panc1 and MiaPaCa2 cells. This dataset contains ozone-induced fatty acid
ST:STUDY_SUMMARY                 	discovery (OzFAD) data, which shows differences in carbon-carbon double bond
ST:STUDY_SUMMARY                 	position between Panc1 and MiaPaCa2 cells. Panc1 cells contain significant
ST:STUDY_SUMMARY                 	amounts of 16:1n-10, which is an unusual isomeric fatty acid produced by Δ6
ST:STUDY_SUMMARY                 	desaturation of palmitic acid by fatty acid desaturase 2 (FADS2). Panc1 GEMR
ST:STUDY_SUMMARY                 	cells show increased stearoyl-CoA desaturase 1 (SCD1)-derived 16:1n-7 alongside
ST:STUDY_SUMMARY                 	decreased 16:1n-10.
ST:INSTITUTE                     	Victor Chang Cardiac Research Institute
ST:LAST_NAME                     	Hancock
ST:FIRST_NAME                    	Sarah
ST:ADDRESS                       	Level 7 Lowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW 2010,
ST:ADDRESS                       	Australia
ST:EMAIL                         	s.hancock@victorchang.edu.au
ST:PHONE                         	+61414537526
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	M64_1	Sample source:MiaPaCa2 cells | group:GEMR	RAW_FILE_NAME(Raw file name)=20240221_MS_5uL_M64_1.mzML
SUBJECT_SAMPLE_FACTORS           	-	M64_2	Sample source:MiaPaCa2 cells | group:GEMR	RAW_FILE_NAME(Raw file name)=20240221_MS_5uL_M64_2.mzML
SUBJECT_SAMPLE_FACTORS           	-	M64_3	Sample source:MiaPaCa2 cells | group:GEMR	RAW_FILE_NAME(Raw file name)=20240221_MS_5uL_M64_3.mzML
SUBJECT_SAMPLE_FACTORS           	-	MIA_1	Sample source:MiaPaCa2 cells | group:CON	RAW_FILE_NAME(Raw file name)=20240221_MS_5uL_MIA_1.mzML
SUBJECT_SAMPLE_FACTORS           	-	MIA_2	Sample source:MiaPaCa2 cells | group:CON	RAW_FILE_NAME(Raw file name)=20240221_MS_5uL_MIA_2.mzML
SUBJECT_SAMPLE_FACTORS           	-	MIA_3	Sample source:MiaPaCa2 cells | group:CON	RAW_FILE_NAME(Raw file name)=20240221_MS_5uL_MIA_3.mzML
SUBJECT_SAMPLE_FACTORS           	-	P150_1	Sample source:Panc1 cells | group:GEMR	RAW_FILE_NAME(Raw file name)=20240221_MS_5uL_P150_1.mzML
SUBJECT_SAMPLE_FACTORS           	-	P150_2	Sample source:Panc1 cells | group:GEMR	RAW_FILE_NAME(Raw file name)=20240221_MS_5uL_P150_2.mzML
SUBJECT_SAMPLE_FACTORS           	-	P150_3	Sample source:Panc1 cells | group:GEMR	RAW_FILE_NAME(Raw file name)=20240221_MS_5uL_P150_3.mzML
SUBJECT_SAMPLE_FACTORS           	-	PANC_1	Sample source:Panc1 cells | group:CON	RAW_FILE_NAME(Raw file name)=20240221_MS_5uL_PANC_1.mzML
SUBJECT_SAMPLE_FACTORS           	-	PANC_2	Sample source:Panc1 cells | group:CON	RAW_FILE_NAME(Raw file name)=20240221_MS_5uL_PANC_2.mzML
SUBJECT_SAMPLE_FACTORS           	-	PANC_3	Sample source:Panc1 cells | group:CON	RAW_FILE_NAME(Raw file name)=20240221_MS_5uL_PANC_3.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	Samples were obtained from established immortalised adherent human pancreatic
CO:COLLECTION_SUMMARY            	ductal adenocarcinoma cells (Panc1). Cells were cultured in high glucose DMEM
CO:COLLECTION_SUMMARY            	containing 4.5 g/L glucose and 4 mM glutamine and were supplemented with 10%
CO:COLLECTION_SUMMARY            	FCS. Cells were cultured under 5% CO2 at 37ºC and routinely passaged every 2-3
CO:COLLECTION_SUMMARY            	days (when ~80-90% confluent). Cell lines were regularly screened for mycoplasma
CO:COLLECTION_SUMMARY            	infection.
CO:SAMPLE_TYPE                   	Pancreas
#TREATMENT
TR:TREATMENT_SUMMARY             	Panc1 GEMR cells were produced by serial treatment with escalating doses of
TR:TREATMENT_SUMMARY             	gemcitabine for approximately 12 weeks. The final concentration of GEMC for
TR:TREATMENT_SUMMARY             	Panc1 cells was 150 nM, resulting in a half-maximal inhibitory concentration of
TR:TREATMENT_SUMMARY             	720.5 nM (approximately 2.5 times increase over control (CON) cells, 273.3 nM).
TR:TREATMENT_SUMMARY             	The final concentration of GEMC for MiaPaCa2 cells was 64 nM, resulting in a
TR:TREATMENT_SUMMARY             	half-maximal inhibitory concentration of 92.2 nM (approximately 3x increase over
TR:TREATMENT_SUMMARY             	control (CON) cells, 30.6 nM).
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Lipids were extracted from cells grown to 80-90% confluency in 6 well plates
SP:SAMPLEPREP_SUMMARY            	using a modified methyl tert-butyl ether (MTBE) method. Adherent cells were
SP:SAMPLEPREP_SUMMARY            	washed with ice-cold PBS and scraped into methanol containing 0.01% butylated
SP:SAMPLEPREP_SUMMARY            	hydroxytoluene and 100 pmol each of internal standards (phosphatidylcholine
SP:SAMPLEPREP_SUMMARY            	19:0/19:0, phosphatidylethanolamine 17:0/17:0, phosphatidylserine 17:0/17:0,
SP:SAMPLEPREP_SUMMARY            	lysophosphatidylcholine 17:0, lysophosphatidylethanolamine 14:0,
SP:SAMPLEPREP_SUMMARY            	phosphatidylglycerol 17:0/17:0, phosphatidic acid 17:0/17:0, ceramide
SP:SAMPLEPREP_SUMMARY            	d18:1/17:0, dihydrosphingomyelin 12:0, cholesteryl ester 22:1, diacylglycerol
SP:SAMPLEPREP_SUMMARY            	17:0/17:0, D5-triacylglycerol 16:0/16:0/16:0 and cardiolipin
SP:SAMPLEPREP_SUMMARY            	14:0/14:0/14:0/14:0, and 20 nmol of methyl nonadecanoate). An empty well was
SP:SAMPLEPREP_SUMMARY            	also scraped for use in background subtraction. To this, MTBE was added at a
SP:SAMPLEPREP_SUMMARY            	ratio of 3:1 v/v, and extracts were rotated overnight at 4ºC. Following
SP:SAMPLEPREP_SUMMARY            	overnight rotation, 1 part of ice-cold 150 mM ammonium acetate was added,
SP:SAMPLEPREP_SUMMARY            	samples were vortexed vigorously and centrifuged at 2000 x g for 5 min. The
SP:SAMPLEPREP_SUMMARY            	upper organic phase containing lipids was transferred into a 1.5 mL autosampler
SP:SAMPLEPREP_SUMMARY            	vial and dried under N2 at 37ºC Dried lipids were reconstituted in
SP:SAMPLEPREP_SUMMARY            	chloroform:methanol:water (60:30:4.5 v/v/v), transferred to a sleeved vial, and
SP:SAMPLEPREP_SUMMARY            	stored at -20ºC until analysis. The aqueous phase containing the protein pellet
SP:SAMPLEPREP_SUMMARY            	was dried under N2 and digested with 1M NaOH overnight at 4ºC. Digested protein
SP:SAMPLEPREP_SUMMARY            	was diluted 1:2 v/v with water and protein concentration was determined using a
SP:SAMPLEPREP_SUMMARY            	Pierce BCA assay kit (ThermoFisher Scientific) Following MTBE extraction of
SP:SAMPLEPREP_SUMMARY            	lipids, an aliquot was taken and hydrolysed in 0.6 M KOH in 75% v/v methanol at
SP:SAMPLEPREP_SUMMARY            	60ºC for 30 min. Samples were then cooled to room temperature and neutralised
SP:SAMPLEPREP_SUMMARY            	with 25% v/v acetic acid. Water and n-hexane (3:2 v/v) were added separately and
SP:SAMPLEPREP_SUMMARY            	samples were vortexed vigorously before being centrifuged at 2000 x g for 5 min.
SP:SAMPLEPREP_SUMMARY            	The upper phase containing hydrolysed fatty acids was removed, and the aqueous
SP:SAMPLEPREP_SUMMARY            	phase was washed with a second volume of n-hexane and combined. The combined
SP:SAMPLEPREP_SUMMARY            	organic phase was then dried under N2 at 37°C. Dried hydrolysed fatty acids
SP:SAMPLEPREP_SUMMARY            	were derivatised with an AMP+ MaxSpec kit (Cayman Chemical, Ann Arbor, MI, USA)
SP:SAMPLEPREP_SUMMARY            	as per manufacturer instructions. Following this, AMP-derivatised fatty acids
SP:SAMPLEPREP_SUMMARY            	were re-extracted using MTBE:water (1:1 v/v), and the upper MTBE phase was dried
SP:SAMPLEPREP_SUMMARY            	under N2 before being resuspended in methanol. Samples were stored at -20°C
SP:SAMPLEPREP_SUMMARY            	until analysis. A 37-component fatty acid methyl ester standard (Merck Life
SP:SAMPLEPREP_SUMMARY            	Sciences) containing an additional 3.184 nmol of methyl nonadecanoate was
SP:SAMPLEPREP_SUMMARY            	concurrently subjected to the same derivatisation procedure and used as an
SP:SAMPLEPREP_SUMMARY            	external quality control for fatty acid identification by LC-MS.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
CH:SOLVENT_A                     	Water (0.1% formic acid)
CH:SOLVENT_B                     	Acetonitrile (0.1% formic acid)
CH:FLOW_GRADIENT                 	0 - 0.5 min at 20% B, then linearly increased to 90% B (16 min), then 100% B
CH:FLOW_GRADIENT                 	(17.35 min), held at 100% B until 20.35 min, reduced to 20% B and held until 21
CH:FLOW_GRADIENT                 	min.
CH:FLOW_RATE                     	0.4 mL/min
CH:COLUMN_TEMPERATURE            	60
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Waters Synapt G2 Si QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Chromatographic detection was conducted using an ion-mobility enabled quadrupole
MS:MS_COMMENTS                   	time of flight (QTOF) mass spectrometer (SYNAPT G2-Si HDMS, Waters Corporation)
MS:MS_COMMENTS                   	previously modified to allow introduction of high concentration ozone into the
MS:MS_COMMENTS                   	ion mobility region of the instrument 87. Electrospray ionisation parameters
MS:MS_COMMENTS                   	were: capillary +3 kV, sampling cone +40 V, source temperature 120 °C,
MS:MS_COMMENTS                   	desolvation temperature 550 °C, cone gas 100 L/h, desolvation gas 900 L/h, and
MS:MS_COMMENTS                   	nebulizer gas pressure 6.5 bar. Gas flows to the ion mobility region of the
MS:MS_COMMENTS                   	instrument were 2.0, 180 and 90 mL/min, for the trap, helium cell and IMS
MS:MS_COMMENTS                   	region, respectively. For the ozonolysis reactions, oxygen (≥ 99.999%,
MS:MS_COMMENTS                   	Coregas, NSW, Australia) was converted to ozone at concentrations of 220-240
MS:MS_COMMENTS                   	g/Nm3 (~15-17% O3 in O2) using a high concentration ozone generator (TG-40,
MS:MS_COMMENTS                   	Ozone Solutions, Hull, IA). Peak areas for Criegee and aldehyde ions for each
MS:MS_COMMENTS                   	monounsaturated fatty acid isomer were combined and normalised to internal
MS:MS_COMMENTS                   	standard and total protein (measured by BCA assay).
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	normalisd peak area
MS_METABOLITE_DATA_START
Samples	M64_1	M64_2	M64_3	MIA_1	MIA_2	MIA_3	P150_1	P150_2	P150_3	PANC_1	PANC_2	PANC_3
Factors	Sample source:MiaPaCa2 cells | group:GEMR	Sample source:MiaPaCa2 cells | group:GEMR	Sample source:MiaPaCa2 cells | group:GEMR	Sample source:MiaPaCa2 cells | group:CON	Sample source:MiaPaCa2 cells | group:CON	Sample source:MiaPaCa2 cells | group:CON	Sample source:Panc1 cells | group:GEMR	Sample source:Panc1 cells | group:GEMR	Sample source:Panc1 cells | group:GEMR	Sample source:Panc1 cells | group:CON	Sample source:Panc1 cells | group:CON	Sample source:Panc1 cells | group:CON
16:1n-7 Precursor	4911440	4433769	4110076	5604789	7088610	7187151	3298023	6133222	6969477	5608628	4380095	6287812
16:1n-7 Criegee	103987	92644	86346	127752	163806	142836	55700	98087	125343	80083	60540	86765
16:1n-7 Aldehyde	188257	176388	159919	257358	325359	290576	107895	210154	245226	160837	124246	176238
16:1n-9 Precursor	4911440	4433769	4110076	5604789	7088610	7187151	3298023	6133222	6969477	5608628	4380095	6287812
16:1n-9 Criegee	28877	25847	27740	14108	16256	11401	14862	12769	15098	19397	13705	17178
16:1n-9 Aldehyde	45014	36210	42391	20367	28263	24914	28263	23371	29955	29918	22287	23158
16:1n-10 Precursor	4911440	4433769	4110076	5604789	7088610	7187151	3298023	6133222	6969477	5608628	4380095	6287812
16:1n-10 Criegee	3250	4731	4602	5005	5326	25808	10118	33569	30292	51441	40517	53449
16:1n-10 Aldehyde	7798	13323	9864	11858	13729	79371	27157	108741	81741	145769	116159	164050
18:1n-7 Precursor	19084702	18049386	17034088	17390728	18739616	19088110	15168191	17350246	20111674	20435610	17986006	21518846
18:1n-7 Criegee	424620	382325	325597	507677	576516	526628	437529	510085	657819	492482	388690	531993
18:1n-7 Aldehyde	714680	652173	539919	847588	981376	856802	654457	863582	1022688	770670	615659	846611
18:1n-9 Precursor	19084702	18049386	17034088	17390728	18739616	19088110	15168191	17350246	20111674	20435610	17986006	21518846
18:1n-9 Criegee	769771	720572	700932	555322	615759	603679	373498	452742	503228	563943	486648	634262
18:1n-9 Aldehyde	1475259	1373542	1337652	1063182	1188364	1161493	715192	895421	971775	1120753	959157	1245847
18:1n-10 Precursor	19084702	18049386	17034088	17390728	18739616	19088110	15168191	17350246	20111674	20435610	17986006	21518846
18:1n-10 Criegee	42204	41154	38135	33938	37257	60592	57453	133776	133828	221776	171641	237602
18:1n-10 Aldehyde	57021	60633	57406	58039	62192	111483	125147	283241	311193	501436	395453	541379
18:1n-12 Precursor	19084702	18049386	17034088	17390728	18739616	19088110	15168191	17350246	20111674	20435610	17986006	21518846
18:1n-12 Criegee	1576	1622	1591	1608	590	4747	2564	8451	3581	15126	10725	14277
18:1n-12 Aldehyde	7335	5796	6147	7290	7706	14699	4341	19962	11301	37613	35378	40973
19:0 Precursor	566186	523165	565939	469620	510180	713276	545260	666036	595391	519332	483600	624449
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	PrecursorMz	ProductMz
16:1n-7 Precursor	421.3814	421.3814
16:1n-7 Criegee	421.3814	355.2679
16:1n-7 Aldehyde	421.3814	339.2787
16:1n-9 Precursor	421.3814	421.3814
16:1n-9 Criegee	421.3814	327.2402
16:1n-9 Aldehyde	421.3814	311.2473
16:1n-10 Precursor	421.3814	421.3814
16:1n-10 Criegee	421.3814	313.2293
16:1n-10 Aldehyde	421.3814	297.2331
18:1n-7 Precursor	449.4	449.4
18:1n-7 Criegee	449.4	383.2926
18:1n-7 Aldehyde	449.4	367.3047
18:1n-9 Precursor	449.4	449.4
18:1n-9 Criegee	449.4	355.2679
18:1n-9 Aldehyde	449.4	339.2787
18:1n-10 Precursor	449.4	449.4
18:1n-10 Criegee	449.4	341.2593
18:1n-10 Aldehyde	449.4	325.2615
18:1n-12 Precursor	449.4	449.4
18:1n-12 Criegee	449.4	313.2293
18:1n-12 Aldehyde	449.4	297.2331
19:0 Precursor	437.4068	437.4068
METABOLITES_END
#END