#METABOLOMICS WORKBENCH kwessendorf_20251205_151122 DATATRACK_ID:6845 STUDY_ID:ST004450 ANALYSIS_ID:AN007453 PROJECT_ID:PR002810
VERSION             	1
CREATED_ON             	December 16, 2025, 9:36 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 Insititute 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 impact of an SMPD1 inhibitor on lipid homeostasis
ST:STUDY_SUMMARY                 	An H1299 cell line was cultured in 10% FBS for 24 hours in high glucose DMEM
ST:STUDY_SUMMARY                 	media containing either either 1µM ARC39, an acid sphingomyelinase inhibitor,
ST:STUDY_SUMMARY                 	or vehicle (0.1% DMSO). At the end of the 24 hours of treatment, wells were
ST:STUDY_SUMMARY                 	washed twice with 0.9% w/V saline, then scraped into 100% methanol. Internal
ST:STUDY_SUMMARY                 	standards were added and samples were split for polar or neutral lipid analysis
ST:STUDY_SUMMARY                 	by liquid chromatography coupled to high-resolution mass spectrometry. MS data
ST:STUDY_SUMMARY                 	was analyzed using EL-Maven v0.12.1 with a mass error of 5ppm and maximum
ST:STUDY_SUMMARY                 	retention time shift between parent ions and isotopomer ions of 5 seconds. An
ST:STUDY_SUMMARY                 	in-house matlab script 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           	H1299 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           	H1299_Veh_1	H1299_Veh_1	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:0.1% v/V DMSO (Vehicle) | Injection order:B3	RAW_FILE_NAME(Raw_File_Name)=H1299_Veh_1.mzXML
SUBJECT_SAMPLE_FACTORS           	H1299_Veh_2	H1299_Veh_2	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:0.1% v/V DMSO (Vehicle) | Injection order:B3	RAW_FILE_NAME(Raw_File_Name)=H1299_Veh_2.mzXML
SUBJECT_SAMPLE_FACTORS           	H1299_Veh_3	H1299_Veh_3	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:0.1% v/V DMSO (Vehicle) | Injection order:B3	RAW_FILE_NAME(Raw_File_Name)=H1299_Veh_3.mzXML
SUBJECT_SAMPLE_FACTORS           	H1299_SMPD1i_1	H1299_SMPD1i_1	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:1µM ARC39 (SMPD1 inhibitor) | Injection order:B3	RAW_FILE_NAME(Raw_File_Name)=H1299_SMPD1i_1.mzXML
SUBJECT_SAMPLE_FACTORS           	H1299_SMPD1i_2	H1299_SMPD1i_2	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:1µM ARC39 (SMPD1 inhibitor) | Injection order:B3	RAW_FILE_NAME(Raw_File_Name)=H1299_SMPD1i_2.mzXML
SUBJECT_SAMPLE_FACTORS           	H1299_SMPD1i_3	H1299_SMPD1i_3	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:1µM ARC39 (SMPD1 inhibitor) | Injection order:B3	RAW_FILE_NAME(Raw_File_Name)=H1299_SMPD1i_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                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	An H1299 cell line was cultured with either vehicle (0.1% DMSO) or 1µM ARC39,
TR:TREATMENT_SUMMARY             	an acid sphingomyelinase inhibitor, and 10% FBS for 24 hours in high glucose.
TR:TREATMENT_SUMMARY             	The goal was to determine the effect of inhibiting acid sphingomyelinase to the
TR:TREATMENT_SUMMARY             	lipidome of H1299 cells.
#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_SUMMARY        	All metabolites abundances were calculated from C18 analysis of cells cultured
CH:CHROMATOGRAPHY_SUMMARY        	in 13C serine containing media except for TG16:0_18:1_18:0 which was acquired
CH:CHROMATOGRAPHY_SUMMARY        	from C30 due to improper peak on C18.
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	H1299_Veh_1	H1299_Veh_2	H1299_Veh_3	H1299_SMPD1i_1	H1299_SMPD1i_2	H1299_SMPD1i_3																								
Factors	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:0.1% v/V DMSO (Vehicle) | Injection order:B3	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:0.1% v/V DMSO (Vehicle) | Injection order:B3	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:0.1% v/V DMSO (Vehicle) | Injection order:B3	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:1µM ARC39 (SMPD1 inhibitor) | Injection order:B3	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:1µM ARC39 (SMPD1 inhibitor) | Injection order:B3	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:1µM ARC39 (SMPD1 inhibitor) | Injection order:B3
SM 32:1;O2	0.590899203	0.533260217	0.682254718	0.716638752	0.649321041	0.726803024																								
SM 33:1;O2	0.332064091	0.307086101	0.427635921	0.46031093	0.387199723	0.419237014																								
SM 34:1;O2	6.328723395	6.697419224	8.066517513	7.821080628	7.416541319	9.055310815																								
SM 36:1;O2	0.204826532	0.199949825	0.196943232	0.536283246	0.595841899	0.567562016																								
SM 40:1;O2	0.271476643	0.236263947	0.262965989	0.885028967	0.780701414	0.88628797																								
SM 40:2;O2	0.112922999	0.082421838	0.092135912	0.278863682	0.34796765	0.337874722																								
SM 42:1;O2	0.953129397	0.859312293	0.991768708	1.353445635	1.654019949	1.207678254																								
SM 42:2;O2	2.174671277	2.155301973	2.263463611	3.495245119	3.345047925	3.362605741																								
HexCer 18:1;O2/24:0	1.971620396	1.915193366	1.819185872	1.856491138	2.056912956	1.856405255																								
HexCer 18:1;O2/24:1	1.250383108	1.368612084	1.174772016	1.303666326	1.257546903	1.182676744																								
Lact-Cer 18:1;O2/16:0	1.183654239	1.143302759	1.304862796	1.192365609	1.055220599	1.028837661																								
Lact-Cer 18:1;O2/24:0	0.769395128	0.761586297	0.851340276	0.694170415	0.707518669	0.669151612																								
Lact-Cer 18:1;O2/24:1	0.750551464	0.722983647	0.776927623	0.747081054	0.706901725	0.680436252																								
GM3 18:1;O2/16:0	2.644352015	2.415938228	2.664030859	2.848624145	2.369013884	2.769382179																								
GM2 18:1;O2/16:0	3.943822915	4.118400675	3.965426605	4.715244455	4.32015138	4.366405074																								
Lc3 18:1;O2/16:0	0.178375722	0.182670873	0.200221415	0.188017829	0.164921674	0.158935916																								
Gb3 18:1;O2/16:0	2.641049928	2.569798847	2.582324622	2.799154667	2.781034325	2.704507193																								
Gb3 18:1;O2/24:0	1.72932072	1.390042828	1.7072457	1.900249639	1.684443726	1.826473184																								
Gb3 18:1;O2/24:1	1.647904603	1.69623674	1.868713585	2.052780524	1.859376586	1.906222812																								
PC 32:0	0.532624302	0.60911192	0.585856379	0.685073093	0.557090422	0.558701426																								
PC 32:1	0.758142635	0.865005549	0.958334585	0.943963134	0.719064605	0.804787064																								
PC 32:2	0.492805441	0.576204656	0.62525258	0.549555669	0.482128223	0.523430063																								
PC 33:1	0.480735882	0.546998476	0.570601456	0.59144357	0.481707815	0.53225724																								
PC 34:0	0.062619712	0.077925422	0.074617972	0.087358573	0.070860056	0.07856996																								
PC 34:1	0.032848969	0.044322204	0.035801933	0.044738032	0.043107307	0.044171811																								
PC 34:2	0.02946298	0.037674771	0.034333601	0.034877354	0.029106725	0.025508865																								
PC 34:3	0.43623202	0.530005588	0.442812286	0.519243884	0.490819664	0.434067462																								
PC 35:1	0.285638322	0.28668799	0.292755359	0.352496778	0.297284394	0.293144933																								
PC 35:2	0.27219052	0.349838647	0.320443575	0.380791295	0.328157975	0.305944573																								
PC 36:1	0.191652621	0.191356546	0.183109978	0.183520338	0.150762392	0.161759943																								
PC 36:2	0.717641484	0.777895796	0.783883931	0.847077394	0.709685938	0.77611646																								
PC 36:3	0.161964149	0.187890116	0.183923512	0.210318286	0.161319142	0.170765947																								
PC 36:4 _1	0.416665902	0.423398186	0.447199953	0.560556789	0.438702014	0.461286627																								
PC 36:4 _2	0.044291493	0.0439013	0.0471474	0.061229368	0.044999248	0.044150826																								
PC 36:5_1	0.282485212	0.329773982	0.369239741	0.41267891	0.313188294	0.352235369																								
PC 36:5_2	0.332947186	0.415111714	0.459666196	0.485268273	0.378800925	0.418206872																								
PC 38:1	0.294166272	0.32545035	0.311791774	0.408498961	0.341770076	0.373187468																								
PC 38:2	0.01377792	0.016532789	0.015663869	0.019607838	0.016738516	0.018073176																								
PC 38:3	0.043582482	0.048382179	0.044935433	0.053489146	0.046422475	0.050441862																								
PC 38:4_1	0.019320196	0.023569941	0.021894096	0.026198205	0.024078942	0.026696011																								
PC 38:4_2	0.007600492	0.009294532	0.009383149	0.01034261	0.009272419	0.010320215																								
PC 38:5	0.013662644	0.01678875	0.015964694	0.017890584	0.01638475	0.018071165																								
PC 38:6	7.165481426	6.877087767	6.484259914	7.026921362	7.295887428	6.835037273																								
PC 38:7	32.66462249	34.89582977	36.21018832	37.57309872	34.06273388	36.83920228																								
PE 32:1	6.761901855	7.886832968	8.249159443	8.653661002	7.766243442	8.865635441																								
PE 34:1	7.483492382	8.388034936	7.464460924	7.931994623	7.746475054	7.872761857																								
PE 34:2	3.159591511	4.21394492	3.747257122	3.415637807	3.318711298	3.734869887																								
PE 36:1	101.1846541	118.3696667	111.837715	116.5853811	110.3861112	110.198584																								
PE 36:2	41.74509806	45.90035281	52.73117813	51.97285571	45.2792231	45.29467038																								
PE 36:3	0.052572157	0.070380439	0.073280488	0.055785789	0.06266471	0.063562648																								
PE 36:4	0.125866901	0.117791368	0.101992516	0.116987474	0.120172563	0.116764923																								
PE 36:5	0.159899677	0.145445334	0.156398109	0.158445936	0.176570509	0.163080984																								
PE 38:2	0.965725295	0.946981624	1.065332661	0.970296271	0.981835441	0.966099757																								
PE 38:3	1.202372642	1.253255138	1.20631461	1.218157202	1.280725963	1.154269045																								
PE 38:4	0.19700737	0.190018078	0.186672587	0.195790916	0.198144243	0.125131325																								
PE 38:5	0.019108256	0.019336915	0.019510085	0.019844867	0.017518371	0.018429514																								
PE 38:6	0.114168133	0.119254511	0.139977211	0.114971486	0.116710237	0.10893072																								
PE 40:1	0.015690867	0.01683211	0.018671945	0.015353647	0.015000071	0.014621253																								
PE 40:4	0.02822778	0.029901095	0.032357323	0.026262059	0.0274169	0.027416256																								
PE 40:5	0.021930187	0.022310374	0.023609135	0.021572598	0.020994452	0.020307403																								
PE 40:6	0.17329131	0.174038733	0.177991093	0.183531689	0.17750963	0.166369184																								
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Retention time	m/z ratio
SM 32:1;O2	10.71	675.5433
SM 33:1;O2	11.02	689.5588
SM 34:1;O2	11.30	703.5746
SM 36:1;O2	11.84	731.6057
SM 40:1;O2	13.00	787.6691
SM 40:2;O2	12.51	785.6532
SM 42:1;O2	13.70	815.7009
SM 42:2;O2	13.04	813.6852
HexCer 18:1;O2/24:0	13.56	812.6984
HexCer 18:1;O2/24:1	12.97	810.6827
Lact-Cer 18:1;O2/16:0	11.09	862.6245
Lact-Cer 18:1;O2/24:0	13.16	974.7509
Lact-Cer 18:1;O2/24:1	12.64	972.7352
GM3 18:1;O2/16:0	9.94	1356.8008
GM2 18:1;O2/16:0	10.01	1153.7213
Lc3 18:1;O2/16:0	10.85	1065.7031
Gb3 18:1;O2/16:0	10.89	1024.6776
Gb3 18:1;O2/24:0	12.90	1136.8040
Gb3 18:1;O2/24:1	12.42	1134.7877
PC 32:0	11.90	734.5695
PC 32:1	11.52	732.5537
PC 32:2	11.09	730.5379
PC 33:1	11.75	746.5696
PC 34:0	12.44	762.6003
PC 34:1	12.00	760.5853
PC 34:2	11.63	758.5694
PC 34:3	11.26	756.5533
PC 35:1	12.27	774.6011
PC 35:2	11.85	772.5856
PC 36:1	12.55	788.6168
PC 36:2	12.11	786.6008
PC 36:3	11.80	784.5849
PC 36:4 _1	11.52	782.5688
PC 36:4 _2	11.65	782.5688
PC 36:5_1	11.24	780.5539
PC 36:5_2	11.36	780.5534
PC 38:1	13.16	816.6483
PC 38:2	12.63	814.6327
PC 38:3	12.36	812.6165
PC 38:4_1	11.93	810.6002
PC 38:4_2	12.17	810.6008
PC 38:5	11.75	808.5848
PC 38:6	11.60	806.5688
PC 38:7	11.19	804.5535
PE 32:1	11.54	690.5065
PE 34:1	12.02	718.5383
PE 34:2	11.63	716.5227
PE 36:1	12.54	746.5701
PE 36:2	12.12	744.5543
PE 36:3	11.81	742.5388
PE 36:4	11.68	740.5229
PE 36:5	11.28	738.5067
PE 38:2	12.61	772.5859
PE 38:3	12.38	770.5696
PE 38:4	12.18	768.5544
PE 38:5	11.78	766.5381
PE 38:6	11.63	764.5228
PE 40:1	13.82	802.6328
PE 40:4	12.57	796.5842
PE 40:5	12.23	794.5698
PE 40:6	12.12	792.5538
METABOLITES_END
#END