#METABOLOMICS WORKBENCH kwessendorf_20251208_153900 DATATRACK_ID:6859 STUDY_ID:ST004460 ANALYSIS_ID:AN007467 PROJECT_ID:PR002810
VERSION             	1
CREATED_ON             	December 17, 2025, 8:15 am
#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
PR:EMAIL                         	kwessendorf@salk.edu
PR:PHONE                         	7874490440
#STUDY
ST:STUDY_TITLE                   	Determine the impact of a moderate concentrations of fumonisin B1 to
ST:STUDY_TITLE                   	sphingolipid abundances and CERS flux
ST:STUDY_SUMMARY                 	An A549 cell line was cultured in 20% FBS for 24 hours in high glucose DMEM
ST:STUDY_SUMMARY                 	media containing 13C serine and 13C glycine and treated with either 2µM, 5µM,
ST:STUDY_SUMMARY                 	or 10µM fumonisin B1, a ceramide synthase inhibitor, or vehicle (0.1% DMSO). At
ST:STUDY_SUMMARY                 	the end of the 24 hours of treatment, wells were washed twice with 0.9% w/V
ST:STUDY_SUMMARY                 	saline, then scraped into 100% methanol. Internal standards were added and
ST:STUDY_SUMMARY                 	samples were split for polar or neutral lipid analysis by liquid chromatography
ST:STUDY_SUMMARY                 	coupled to high-resolution mass spectrometry. MS data was analyzed using
ST:STUDY_SUMMARY                 	EL-Maven v0.12.1 with a mass error of 5ppm and maximum retention time shift
ST:STUDY_SUMMARY                 	between parent ions and isotopomer ions of 5 seconds. An in-house matlab script
ST:STUDY_SUMMARY                 	was used for natural 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           	DMSO_1	DMSO_1	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]serine | Injection order:B6	RAW_FILE_NAME(Raw_File_Name)=DMSO_1.mzXML
SUBJECT_SAMPLE_FACTORS           	DMSO_2	DMSO_2	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]serine | Injection order:B6	RAW_FILE_NAME(Raw_File_Name)=DMSO_2.mzXML
SUBJECT_SAMPLE_FACTORS           	DMSO_3	DMSO_3	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]serine | Injection order:B6	RAW_FILE_NAME(Raw_File_Name)=DMSO_3.mzXML
SUBJECT_SAMPLE_FACTORS           	FumB1_2µM_1	FumB1_2µM_1	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 2µM Fumonisin B1, [U-13C]serine | Injection order:B6	RAW_FILE_NAME(Raw_File_Name)=FumB1_2uM_1.mzXML
SUBJECT_SAMPLE_FACTORS           	FumB1_2µM_2	FumB1_2µM_2	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 2µM Fumonisin B1, [U-13C]serine | Injection order:B6	RAW_FILE_NAME(Raw_File_Name)=FumB1_2uM_2.mzXML
SUBJECT_SAMPLE_FACTORS           	FumB1_2µM_3	FumB1_2µM_3	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 2µM Fumonisin B1, [U-13C]serine | Injection order:B6	RAW_FILE_NAME(Raw_File_Name)=FumB1_2uM_3.mzXML
SUBJECT_SAMPLE_FACTORS           	FumB1_5µM_1	FumB1_5µM_1	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 5µM Fumonisin B1, [U-13C]serine | Injection order:B6	RAW_FILE_NAME(Raw_File_Name)=FumB1_5uM_1.mzXML
SUBJECT_SAMPLE_FACTORS           	FumB1_5µM_2	FumB1_5µM_2	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 5µM Fumonisin B1, [U-13C]serine | Injection order:B6	RAW_FILE_NAME(Raw_File_Name)=FumB1_5uM_2.mzXML
SUBJECT_SAMPLE_FACTORS           	FumB1_5µM_3	FumB1_5µM_3	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 5µM Fumonisin B1, [U-13C]serine | Injection order:B6	RAW_FILE_NAME(Raw_File_Name)=FumB1_5uM_3.mzXML
SUBJECT_SAMPLE_FACTORS           	FumB1_10µM_1	FumB1_10µM_1	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 10µM Fumonisin B1, [U-13C]serine | Injection order:B6	RAW_FILE_NAME(Raw_File_Name)=FumB1_10uM_1.mzXML
SUBJECT_SAMPLE_FACTORS           	FumB1_10µM_2	FumB1_10µM_2	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 10µM Fumonisin B1, [U-13C]serine | Injection order:B6	RAW_FILE_NAME(Raw_File_Name)=FumB1_10uM_2.mzXML
SUBJECT_SAMPLE_FACTORS           	FumB1_10µM_3	FumB1_10µM_3	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 10µM Fumonisin B1, [U-13C]serine | Injection order:B6	RAW_FILE_NAME(Raw_File_Name)=FumB1_10uM_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 in vehicle (0.1% DMSO) or one of 2µM, 5µM, and 10µM
TR:TREATMENT_SUMMARY             	fumonisin B1 for 24 hours in high glucose media supplemented with 20% FBS and
TR:TREATMENT_SUMMARY             	containing 13C serine and 13C glycine.
#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	DMSO_1	DMSO_2	DMSO_3	FumB1_2µM_1	FumB1_2µM_2	FumB1_2µM_3	FumB1_5µM_1	FumB1_5µM_2	FumB1_5µM_3	FumB1_10µM_1	FumB1_10µM_2	FumB1_10µM_3
Factors	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]serine | Injection order:B6	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]serine | Injection order:B6	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, Vehicle, [U-13C]serine | Injection order:B6	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 2µM Fumonisin B1, [U-13C]serine | Injection order:B6	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 2µM Fumonisin B1, [U-13C]serine | Injection order:B6	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 2µM Fumonisin B1, [U-13C]serine | Injection order:B6	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 5µM Fumonisin B1, [U-13C]serine | Injection order:B6	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 5µM Fumonisin B1, [U-13C]serine | Injection order:B6	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 5µM Fumonisin B1, [U-13C]serine | Injection order:B6	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 10µM Fumonisin B1, [U-13C]serine | Injection order:B6	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 10µM Fumonisin B1, [U-13C]serine | Injection order:B6	Sample source:A549 Cells | Genotype:WT | Treatment:20% FBS, 10µM Fumonisin B1, [U-13C]serine | Injection order:B6
SPB 18:0;O2	0.0239	0.0191	0.0207	0.107	0.104	0.11	0.254	0.25	0.258	1.97	1.98	1.91
SPB 18:1;O2	0.0465	0.0468	0.049	0.0575	0.0587	0.06	0.0613	0.0602	0.0689	0.0963	0.0946	0.0878
Cer 18:0;O2/16:0	0.027821945	0.027421494	0.022006567	0.013693527	0.013457218	0.013293697	0.009898526	0.003089625	0.006890174	ND	0.000293794	ND
Cer 18:0;O2/22:0	0.014122363	0.016664337	0.014482588	0.020888363	0.021379185	0.019874917	0.020681999	0.019739078	0.015342165	0.009273497	0.010855667	0.010450928
Cer 18:0;O2/24:0	0.058600277	0.057430855	0.055679277	0.072116952	0.073205259	0.071441828	0.070479917	0.068567398	0.062150649	0.043870434	0.047944678	0.045832242
Cer 18:1;O2/16:0	0.07633	0.07229	0.07082	0.04128	0.04935	0.03765	0.02051	0.0196	0.02046	0.00248	0.00103	0.00036
Cer 18:1;O2/22:0	0.04325	0.04399	0.04561	0.05042	0.05478	0.05356	0.04697	0.04786	0.0468	0.02389	0.0225	0.02193
Cer 18:1;O2/23:0	0.01825	0.0174	0.01883	0.01804	0.02201	0.0201	0.01901	0.01727	0.01839	0.0066	0.00752	0.00731
Cer 18:1;O2/24:0	0.52367	0.5102	0.53414	0.56206	0.65855	0.59727	0.54532	0.53996	0.53477	0.25804	0.27352	0.26616
Cer 18:1;O2/24:1	0.19789	0.19484	0.20138	0.20037	0.21237	0.19711	0.19459	0.18905	0.2017	0.09281	0.08711	0.08648
SM 32:0;O2	0.043993823	0.043536989	0.043345795	0.051066729	0.056593908	0.050678424	0.045288981	0.046959063	0.045412838	0.028936188	0.028755745	0.030888861
SM 32:1;O2	0.105282992	0.109656656	0.116580387	0.118033253	0.136507285	0.125758357	0.099326589	0.109285958	0.102272863	0.052511851	0.050854083	0.050755502
SM 33:1;O2	0.0359068	0.034945106	0.036208585	0.030030318	0.03253954	0.027748358	0.024473762	0.025049622	0.026253878	0.012976064	0.019375965	0.019678243
SM 34:0;O2	1.325155377	1.281428286	1.311665544	1.296291223	1.36313525	1.288725814	1.036439713	1.083019038	1.177677139	0.729942878	0.770949196	0.702926664
SM 34:1;O2	3.926917839	3.856216663	4.069167787	3.54605046	3.930786973	3.655868438	3.049340553	2.914951797	3.244101929	1.747990139	1.846298913	1.709232313
SM 35:1;O2	0.049540131	0.046616503	0.044752009	0.047536228	0.046801156	0.053782108	0.033289942	0.033515824	0.03631933	0.016983172	0.019631254	0.023746837
SM 36:1;O2	0.163933156	0.142581997	0.142884275	0.199232677	0.215008518	0.202521764	0.194691774	0.191348981	0.201833804	0.177039587	0.170323559	0.174986761
SM 36:2;O2	0.010362883	0.010592395	0.006271932	0.01171168	0.028637897	0.010051037	0.007622588	0.009819768	0.013506251	0.005134569	0.012352164	0.006465643
SM 38:1;O2	0.0812003	0.0870564	0.0949386	0.1063497	0.1325976	0.116064	0.0958829	0.1007165	0.0992763	0.0856271	0.0856183	0.0902142
SM 40:0;O2	0.129300772	0.134248155	0.135542235	0.182460145	0.195782171	0.178781988	0.176474634	0.185579224	0.161402478	0.179061486	0.153723467	0.162948316
SM 40:1;O2	1.070387991	1.08143859	1.141812129	1.439994339	1.646652246	1.503507291	1.444072767	1.417276241	1.368628963	1.218796254	1.181863037	1.143517621
SM 40:2;O2	0.089784155	0.093349433	0.09316924	0.123219254	0.128035812	0.118456363	0.112095173	0.120794286	0.110913534	0.093225143	0.096807546	0.09377082
SM 41:1;O2	0.300275704	0.300721511	0.297263103	0.395468695	0.435745405	0.407730621	0.379207492	0.386863247	0.36788383	0.388155013	0.382002878	0.381815596
SM 42:0;O2	0.105539777	0.106105439	0.113201537	0.138535154	0.148956071	0.135882085	0.135007497	0.126844662	0.131618968	0.16897018	0.178446063	0.19302765
SM 42:1;O2	2.634446868	2.624819432	2.703765202	3.439227565	3.945819036	3.637295161	3.502306746	3.455530569	3.323347675	3.690106468	3.778706773	3.622899096
SM 42:2;O2	4.277004484	4.213721969	4.385889923	5.235035049	5.932472047	5.467222848	5.110984723	5.071019675	5.20612841	4.810912509	4.959256481	4.765966336
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Retention time	m/z ratio
SPB 18:0;O2	7.19	302.3056
SPB 18:1;O2	7.01	300.2900
Cer 18:0;O2/16:0	12.02	540.5352
Cer 18:0;O2/22:0	13.93	624.6301
Cer 18:0;O2/24:0	14.82	652.6613
Cer 18:1;O2/16:0	11.80	538.5195
Cer 18:1;O2/22:0	13.66	622.6141
Cer 18:1;O2/23:0	14.06	636.6303
Cer 18:1;O2/24:0	14.48	650.6458
Cer 18:1;O2/24:1	13.74	648.6299
SM 32:0;O2	10.90	677.5594
SM 32:1;O2	10.65	675.5439
SM 33:1;O2	10.96	689.5594
SM 34:0;O2	11.46	705.5909
SM 34:1;O2	11.24	703.5754
SM 35:1;O2	11.52	717.5910
SM 36:1;O2	11.79	731.6071
SM 36:2;O2	11.39	729.5914
SM 38:1;O2	12.35	759.6383
SM 40:0;O2	13.20	789.6847
SM 40:1;O2	12.94	787.6703
SM 40:2;O2	12.42	785.6546
SM 41:1;O2	13.27	801.6862
SM 42:0;O2	13.93	817.7126
SM 42:1;O2	13.63	815.7019
SM 42:2;O2	12.99	813.6865
METABOLITES_END
#END