#METABOLOMICS WORKBENCH kwessendorf_20251208_114914 DATATRACK_ID:6858 STUDY_ID:ST004458 ANALYSIS_ID:AN007464 PROJECT_ID:PR002810
VERSION             	1
CREATED_ON             	December 16, 2025, 9:47 pm
#PROJECT
PR:PROJECT_TITLE                 	Modeling Lipid Homeostasis Using Stable Isotope Tracing and Flux Analysis
PR:PROJECT_SUMMARY               	Lipids represent the most diverse pool of metabolites found in cells,
PR:PROJECT_SUMMARY               	facilitating compartmentation, signaling, and other functions. Dysregulation of
PR:PROJECT_SUMMARY               	lipid metabolism is linked to disease states ranging from cancer to
PR:PROJECT_SUMMARY               	neurodegeneration. However, limited tools are available for quantifying
PR:PROJECT_SUMMARY               	metabolic fluxes across the lipidome. To directly measure reaction fluxes
PR:PROJECT_SUMMARY               	encompassing membrane lipid homeostasis, we apply stable isotope tracing, liquid
PR:PROJECT_SUMMARY               	chromatography-high-resolution mass spectrometry, and network-based mass
PR:PROJECT_SUMMARY               	isotopomer modeling to non-small cell lung cancer (NSCLC) models. Lipid
PR:PROJECT_SUMMARY               	metabolic flux analysis (MFA) enables the concurrent quantitation of fatty acid
PR:PROJECT_SUMMARY               	synthesis, elongation, headgroup assembly, and salvage reactions within
PR:PROJECT_SUMMARY               	virtually any biological system. Lipid-MFA highlights distinct changes in fatty
PR:PROJECT_SUMMARY               	acid synthase and very long-chain fatty acid (VLCFA) elongation fluxes in
PR:PROJECT_SUMMARY               	typical culture conditions. Using this approach, we resolve differences in
PR:PROJECT_SUMMARY               	sphingolipid recycling in p53-deficient versus liver kinase B1 (LKB1)-deficient
PR:PROJECT_SUMMARY               	NSCLC tumors using precision-cut lung slice culture. Finally, Lipid-MFA
PR:PROJECT_SUMMARY               	demonstrates the unique trafficking of long-chain versus very long-chain
PR:PROJECT_SUMMARY               	ceramide fluxes as well as the isozyme specificity of a classical ceramide
PR:PROJECT_SUMMARY               	synthase inhibitor. These results illustrate the ability of Lipid-MFA to
PR:PROJECT_SUMMARY               	quantify lipid homeostasis and elucidate molecular mechanisms in membrane lipid
PR:PROJECT_SUMMARY               	metabolism.
PR:INSTITUTE                     	Salk Institute for Biological Studies
PR:LAST_NAME                     	Wessendorf-Rodriguez
PR:FIRST_NAME                    	Karl
PR:ADDRESS                       	10010N Torrey Pines Rd, La Jolla, California, 92037, USA
PR:EMAIL                         	kwessendorf@salk.edu
PR:PHONE                         	7874490440
#STUDY
ST:STUDY_TITLE                   	Determine the effect of a moderate dose of fumonisin B1 to the lipidome of
ST:STUDY_TITLE                   	non-small cell lung cancer cells
ST:STUDY_SUMMARY                 	An A549 cell line was cultured in 20% FBS for 24 hours in high glucose DMEM
ST:STUDY_SUMMARY                 	media containing either 13C glucose or 13C serine and treated with either
ST:STUDY_SUMMARY                 	vehicle (0.1% DMSO) or 2µM fumonisin B1, a ceramide synthase inhibitor. At the
ST:STUDY_SUMMARY                 	end of the 24 hours of treatment, wells were washed twice with 0.9% w/V saline,
ST:STUDY_SUMMARY                 	then scraped into 100% methanol. Internal standards were added and samples were
ST:STUDY_SUMMARY                 	split for polar or neutral lipid analysis by liquid chromatography coupled to
ST:STUDY_SUMMARY                 	high-resolution mass spectrometry. MS data was analyzed using EL-Maven v0.12.1
ST:STUDY_SUMMARY                 	with a mass error of 5ppm and maximum retention time shift between parent ions
ST:STUDY_SUMMARY                 	and isotopomer ions of 5 seconds. An in-house matlab script was used for natural
ST:STUDY_SUMMARY                 	abundance correction.
ST:INSTITUTE                     	Salk Institute for Biological Studies
ST:LAST_NAME                     	Wessendorf-Rodriguez
ST:FIRST_NAME                    	Karl
ST:ADDRESS                       	10010N Torrey Pines Rd, La Jolla, California, 92037, USA
ST:EMAIL                         	kwessendorf@salk.edu
ST:PHONE                         	7874490440
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:CELL_STRAIN_DETAILS           	A549 WT
#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           	A549_Veh_Gluc_1	A549_Veh_Gluc_1	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]glucose | Injection order:B5	RAW_FILE_NAME(Raw_File_Name)=A549_Veh_Gluc_1.mzXML
SUBJECT_SAMPLE_FACTORS           	A549_Veh_Gluc_2	A549_Veh_Gluc_2	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]glucose | Injection order:B5	RAW_FILE_NAME(Raw_File_Name)=A549_Veh_Gluc_2.mzXML
SUBJECT_SAMPLE_FACTORS           	A549_Veh_Gluc_3	A549_Veh_Gluc_3	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]glucose | Injection order:B5	RAW_FILE_NAME(Raw_File_Name)=A549_Veh_Gluc_3.mzXML
SUBJECT_SAMPLE_FACTORS           	A549_Veh_Ser_1	A549_Veh_Ser_1	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]serine | Injection order:B5	RAW_FILE_NAME(Raw_File_Name)=A549_Veh_Ser_1.mzXML
SUBJECT_SAMPLE_FACTORS           	A549_Veh_Ser_2	A549_Veh_Ser_2	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]serine | Injection order:B5	RAW_FILE_NAME(Raw_File_Name)=A549_Veh_Ser_2.mzXML
SUBJECT_SAMPLE_FACTORS           	A549_Veh_Ser_3	A549_Veh_Ser_3	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]serine | Injection order:B5	RAW_FILE_NAME(Raw_File_Name)=A549_Veh_Ser_3.mzXML
SUBJECT_SAMPLE_FACTORS           	A549_FuB1_Gluc_1	A549_FuB1_Gluc_1	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, 2µM FuB1, [U-13C]glucose | Injection order:B5	RAW_FILE_NAME(Raw_File_Name)=A549_FuB1_Gluc_1.mzXML
SUBJECT_SAMPLE_FACTORS           	A549_FuB1_Gluc_2	A549_FuB1_Gluc_2	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, 2µM FuB1, [U-13C]glucose | Injection order:B5	RAW_FILE_NAME(Raw_File_Name)=A549_FuB1_Gluc_2.mzXML
SUBJECT_SAMPLE_FACTORS           	A549_FuB1_Gluc_3	A549_FuB1_Gluc_3	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, 2µM FuB1, [U-13C]glucose | Injection order:B5	RAW_FILE_NAME(Raw_File_Name)=A549_FuB1_Gluc_3.mzXML
SUBJECT_SAMPLE_FACTORS           	A549_FuB1_Ser_1	A549_FuB1_Ser_1	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, 2µM FuB1, [U-13C]serine | Injection order:B5	RAW_FILE_NAME(Raw_File_Name)=A549_FuB1_Ser_1.mzXML
SUBJECT_SAMPLE_FACTORS           	A549_FuB1_Ser_2	A549_FuB1_Ser_2	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, 2µM FuB1, [U-13C]serine | Injection order:B5	RAW_FILE_NAME(Raw_File_Name)=A549_FuB1_Ser_2.mzXML
SUBJECT_SAMPLE_FACTORS           	A549_FuB1_Ser_3	A549_FuB1_Ser_3	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, 2µM FuB1, [U-13C]serine | Injection order:B5	RAW_FILE_NAME(Raw_File_Name)=A549_FuB1_Ser_3.mzXML
#COLLECTION
CO:COLLECTION_SUMMARY            	At the conclusion of the tracing experiment, media was aspirated. Then, cells
CO:COLLECTION_SUMMARY            	were rinsed twice with 0.9% saline solution, then scraped and lysed into 500 μL
CO:COLLECTION_SUMMARY            	of ice-cold methanol. 10 nanomoles of norvaline was added to each sample along
CO:COLLECTION_SUMMARY            	with a mixture of Equisplash (Avanti Polar Lipids, Cat# 330731), and SPB 18:0;O2
CO:COLLECTION_SUMMARY            	[D7] (Avanti Polar Lipids, Cat# 860658), SPB 18:1;O2 [D7] (Avanti Polar Lipids,
CO:COLLECTION_SUMMARY            	Cat# 860657), glucosylceramide 18:1;O2[D7]-15:0 (Avanti Polar Lipids, Cat#
CO:COLLECTION_SUMMARY            	330729), lactosylceramide 18:1;O2[D7] (Avanti Polar Lipids, Cat# 330727), and
CO:COLLECTION_SUMMARY            	GM3-d3 18:1;O2/18:0 [D3] (Cayman Chemicals, item No. 39226). Ten percent of the
CO:COLLECTION_SUMMARY            	lysate was transferred to 96 well plate, dried and redissolved in 20 μL of
CO:COLLECTION_SUMMARY            	M-PER buffer (Thermo Fisher Scientific Cat. No. 78501) for protein estimation
CO:COLLECTION_SUMMARY            	with BCA assay. The 100% methanol used for polar lipid analysis on C18 was
CO:COLLECTION_SUMMARY            	centrifuged at 21,000 g for 15 min at 4°C. 300 μL of methanol were collected
CO:COLLECTION_SUMMARY            	and evaporated under nitrogen and stored at -80°C until resuspended for
CO:COLLECTION_SUMMARY            	analysis.
CO:SAMPLE_TYPE                   	Lung cancer cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Cells were cultured overnight in growth media containing 20% FBS and either
TR:TREATMENT_SUMMARY             	vehicle (0.1% DMSO v/V) or 2µM fumonisin B1. The following morning, cells were
TR:TREATMENT_SUMMARY             	cultured in high glucose DMEM supplemented with 20% FBS, and containing either
TR:TREATMENT_SUMMARY             	13C glucose or 13C serine. After 24 hours of culture, cells were washed with
TR:TREATMENT_SUMMARY             	0.9% w/V sodium chloride and extracted using 100% methanol.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Lipids were extracted from cell pellets using 100% methanol for polar lipids on
SP:SAMPLEPREP_SUMMARY            	a C18 column. A mixture of EquiSplash (Avanti Polar Lipids, Cat#330731) and SPB
SP:SAMPLEPREP_SUMMARY            	18:1;O2 [D7] (Avanti Polar Lipids, Cat# 860657), glucosylceramide
SP:SAMPLEPREP_SUMMARY            	18:1;O2[D7]-15:0 (Avanti Polar Lipids, Cat# 330729), lactosylceramide
SP:SAMPLEPREP_SUMMARY            	18:1;O2[D7] (Avanti Polar Lipids, Cat# 330727), and GM3-d3 18:1;O2/18:0 [D3]
SP:SAMPLEPREP_SUMMARY            	(Cayman Chemicals, item No. 39226) was used for internal standards. Lipid
SP:SAMPLEPREP_SUMMARY            	containing phases were dried under nitrogen and stored at -80C until they were
SP:SAMPLEPREP_SUMMARY            	resuspended for the appropriate analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
CH:SOLVENT_A                     	98:2 v/v water: methanol with 5 mM ammonium acetate
CH:SOLVENT_B                     	50:50 v/v methanol: isopropanol with 5 mM ammonium acetate
CH:FLOW_GRADIENT                 	0 min, 30% B; 1 min, 30% B; 2 min, 70% B; 11 min, 95%B; 17 min, 30%B; 21.5 min,
CH:FLOW_GRADIENT                 	30%B; 27 min, 30% B
CH:FLOW_RATE                     	0.2 mL/min
CH:COLUMN_TEMPERATURE            	35
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Lipids were analyzed in positive mode using spray voltage 3.5 kV. Sweep gas
MS:MS_COMMENTS                   	flow was 1 arbitrary units, auxiliary gas flow 10 arbitrary units and sheath gas
MS:MS_COMMENTS                   	flow 50 arbitrary units, with a capillary temperature of 325 °C. Full mass
MS:MS_COMMENTS                   	spectrometry (scan range 220–2,500 m/z) was used at 140,000 resolution with
MS:MS_COMMENTS                   	10E6 automatic gain control and a maximum injection time of 100 ms. Data were
MS:MS_COMMENTS                   	analyzed using EI-Maven. Mass error was set to 5 ppm for metabolite
MS:MS_COMMENTS                   	identification. Isotopologues were filtered to elute within 5 seconds of the
MS:MS_COMMENTS                   	unlabeled parent ion to ensure all labeled species came from the same
MS:MS_COMMENTS                   	metabolite. Mass isotopologue distributions were analyzed with an in-house
MS:MS_COMMENTS                   	MATLAB script which integrated the metabolite fragment ions and corrected for
MS:MS_COMMENTS                   	natural isotope abundances.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	pmoles/ug protein
MS_METABOLITE_DATA_START
Samples	A549_Veh_Gluc_1	A549_Veh_Gluc_2	A549_Veh_Gluc_3	A549_Veh_Ser_1	A549_Veh_Ser_2	A549_Veh_Ser_3	A549_FuB1_Gluc_1	A549_FuB1_Gluc_2	A549_FuB1_Gluc_3	A549_FuB1_Ser_1	A549_FuB1_Ser_2	A549_FuB1_Ser_3
Factors	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]glucose | Injection order:B5	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]glucose | Injection order:B5	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]glucose | Injection order:B5	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]serine | Injection order:B5	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]serine | Injection order:B5	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]serine | Injection order:B5	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, 2µM FuB1, [U-13C]glucose | Injection order:B5	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, 2µM FuB1, [U-13C]glucose | Injection order:B5	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, 2µM FuB1, [U-13C]glucose | Injection order:B5	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, 2µM FuB1, [U-13C]serine | Injection order:B5	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, 2µM FuB1, [U-13C]serine | Injection order:B5	Sample source:Cultured Cell line | Genotype:WT | Treatment:20% FBS, 2µM FuB1, [U-13C]serine | Injection order:B5
SPB 18:0;O2	N.D	N.D	N.D	0.035	0.037	0.058	N.D	N.D	N.D	1.180	0.860	1.040
SPB 18:1;O2	N.D	N.D	N.D	0.149	0.181	0.214	N.D	N.D	N.D	0.249	0.267	0.262
Cer 18:0;O2/16:0	N.D	N.D	N.D	0.022	0.022	0.048	N.D	N.D	N.D	0.022	0.009	0.008
Cer 18:1;O2/16:0	N.D	N.D	N.D	0.503	0.486	0.624	N.D	N.D	N.D	0.133	0.106	0.124
Cer 18:0;O2/24:0	N.D	N.D	N.D	0.233	0.279	0.281	N.D	N.D	N.D	0.450	0.401	0.449
Cer 18:1;O2/24:0	N.D	N.D	N.D	2.230	2.550	2.490	N.D	N.D	N.D	2.760	2.470	2.550
Cer 18:0;O2/24:1	N.D	N.D	N.D	0.100	0.114	0.141	N.D	N.D	N.D	0.241	0.189	0.177
Cer 18:1;O2/24:1	N.D	N.D	N.D	0.935	0.911	0.968	N.D	N.D	N.D	0.903	0.780	0.833
SM 34:0;O2	N.D	N.D	N.D	8.930	9.800	10.000	N.D	N.D	N.D	11.200	9.880	10.300
SM 34:1;O2	N.D	N.D	N.D	0.291	0.379	0.039	N.D	N.D	N.D	1.000	N.D	1.050
SM 42:0;O2	N.D	N.D	N.D	52.600	60.000	62.000	N.D	N.D	N.D	49.000	41.900	43.400
SM 42:1;O2	N.D	N.D	N.D	8.760	10.100	9.400	N.D	N.D	N.D	18.200	15.000	16.400
SM 42:2;O2	N.D	N.D	N.D	21.200	23.500	22.100	N.D	N.D	N.D	33.800	29.400	30.200
GM2 18:1;O2/16:0	N.D	N.D	N.D	2.669	2.778	2.791	N.D	N.D	N.D	2.518	2.160	2.271
GM2 18:1;O2/24:0	N.D	N.D	N.D	9.967	10.936	11.352	N.D	N.D	N.D	14.298	11.984	12.017
DG 16:0_16:0	N.D	N.D	N.D	0.197	0.298	0.206	N.D	N.D	N.D	0.365	0.231	0.235
DG 18:0_18:1	N.D	N.D	N.D	0.441	0.607	0.631	N.D	N.D	N.D	0.678	0.585	0.545
DG 16:0_20:4	N.D	N.D	N.D	0.189	0.261	0.285	N.D	N.D	N.D	0.304	0.281	0.271
PC 32:0	N.D	N.D	N.D	25.700	29.200	28.900	N.D	N.D	N.D	34.600	30.200	32.400
PC 34:1	N.D	N.D	N.D	311.000	343.000	348.000	N.D	N.D	N.D	362.000	315.000	325.000
PC 36:1	N.D	N.D	N.D	81.700	92.000	91.500	N.D	N.D	N.D	107.000	90.500	93.700
PC 36:4	N.D	N.D	N.D	55.200	46.100	41.300	N.D	N.D	N.D	46.800	47.400	41.500
PE 34:1	N.D	N.D	N.D	3.360	3.910	4.490	N.D	N.D	N.D	4.230	3.720	3.720
PE 36:1	N.D	N.D	N.D	8.450	10.100	7.790	N.D	N.D	N.D	10.700	9.910	10.800
PE 36:4	N.D	N.D	N.D	9.730	8.140	6.850	N.D	N.D	N.D	7.830	7.800	7.170
PS 34:1	N.D	N.D	N.D	4.830	4.850	3.380	N.D	N.D	N.D	1.970	1.700	1.700
PS 36:1	N.D	N.D	N.D	16.900	20.600	15.400	N.D	N.D	N.D	12.600	9.960	9.630
pPE 36:2	N.D	N.D	N.D	6.060	5.070	3.900	N.D	N.D	N.D	5.340	5.180	4.520
pPE 36:5	N.D	N.D	N.D	81.300	68.900	60.400	N.D	N.D	N.D	67.900	66.900	63.700
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Retention time	m/z ratio
SPB 18:0;O2	7.32	302.3064
SPB 18:1;O2	7.11	300.2907
Cer 18:0;O2/16:0	12.34	540.5371
Cer 18:1;O2/16:0	12.12	538.5213
Cer 18:0;O2/24:0	15.50	652.6630
Cer 18:1;O2/24:0	15.11	650.6474
Cer 18:0;O2/24:1	14.59	650.6478
Cer 18:1;O2/24:1	14.26	648.6316
SM 34:0;O2	11.77	705.5930
SM 34:1;O2	11.55	703.5776
SM 42:0;O2	14.48	817.7183
SM 42:1;O2	14.10	815.7037
SM 42:2;O2	13.38	813.6882
GM2 18:1;O2/16:0	10.24	1356.8052
GM2 18:1;O2/24:0	12.14	1468.9303
DG 16:0_16:0	13.49	586.5430
DG 18:0_18:1	13.64	612.5589
DG 16:0_20:4	13.10	634.5435
PC 32:0	12.14	734.5722
PC 34:1	12.25	760.5881
PC 36:1	12.84	788.6201
PC 36:4	11.89	782.5721
PE 34:1	12.29	718.5411
PE 36:1	12.85	746.5733
PE 36:4	11.92	740.5256
PS 34:1	11.40	762.5303
PS 36:1	11.80	790.5617
pPE 36:2	13.21	730.5779
pPE 36:5	12.19	724.5305
METABOLITES_END
#END