#METABOLOMICS WORKBENCH kwessendorf_20251204_112733 DATATRACK_ID:6840 STUDY_ID:ST004449 ANALYSIS_ID:AN007452 PROJECT_ID:PR002810
VERSION             	1
CREATED_ON             	December 16, 2025, 9:23 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                   	Compare lipid homeostasis in two distinct non-small cell lung cancer cell lines
ST:STUDY_SUMMARY                 	An A549 cell line and an H1299 cell line were cultured in 10% FBS for 24 hours
ST:STUDY_SUMMARY                 	in high glucose DMEM media containing either 13C glucose or 13C serine. Tracing
ST:STUDY_SUMMARY                 	experiments were performed in parallel wells to limit variability due to plating
ST:STUDY_SUMMARY                 	or passage number accumulation. At the end of the tracing period, 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
#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_G1	A549_Gluc_1	Sample source:A549 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C6] glucose	Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_Gluc_1.mzMXL
SUBJECT_SAMPLE_FACTORS           	A549_G2	A549_Gluc_2	Sample source:A549 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C6] glucose	Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_Gluc_2.mzMXL
SUBJECT_SAMPLE_FACTORS           	A549_G3	A549_Gluc_3	Sample source:A549 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C6] glucose	Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_Gluc_3.mzMXL
SUBJECT_SAMPLE_FACTORS           	A549_S1	A549_Ser_1	Sample source:A549 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C3] serine	Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_Ser_1.mzMXL
SUBJECT_SAMPLE_FACTORS           	A549_S2	A549_Ser_2	Sample source:A549 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C3] serine	Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_Ser_2.mzMXL
SUBJECT_SAMPLE_FACTORS           	A549_S3	A549_Ser_3	Sample source:A549 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C3] serine	Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_Ser_3.mzMXL
SUBJECT_SAMPLE_FACTORS           	H1299_G1	H1299_Gluc_1	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C6] glucose	Batch=B2; RAW_FILE_NAME(Raw_File_Name)=H1299_Gluc_1.mzMXL
SUBJECT_SAMPLE_FACTORS           	H1299_G2	H1299_Gluc_2	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C6] glucose	Batch=B2; RAW_FILE_NAME(Raw_File_Name)=H1299_Gluc_2.mzMXL
SUBJECT_SAMPLE_FACTORS           	H1299_G3	H1299_Gluc_3	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C6] glucose	Batch=B2; RAW_FILE_NAME(Raw_File_Name)=H1299_Gluc_3.mzMXL
SUBJECT_SAMPLE_FACTORS           	H1299_S1	H1299_Ser_1	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C3] serine	Batch=B2; RAW_FILE_NAME(Raw_File_Name)=H1299_Ser_1.mzMXL
SUBJECT_SAMPLE_FACTORS           	H1299_S2	H1299_Ser_2	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C3] serine	Batch=B2; RAW_FILE_NAME(Raw_File_Name)=H1299_Ser_2.mzMXL
SUBJECT_SAMPLE_FACTORS           	H1299_S3	H1299_Ser_3	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C3] serine	Batch=B2; RAW_FILE_NAME(Raw_File_Name)=H1299_Ser_3.mzMXL
#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             	Prior to changing cells to tracing media, cells were washed with PBS twice.
TR:TREATMENT_SUMMARY             	Then, cells were cultured in high glucose DMEM containing either 13C glucose or
TR:TREATMENT_SUMMARY             	13C serine and supplemented with 10% FBS for 24 hours.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Lipids were extracted from cell pellets using 100% methanol for polar lipids on
SP:SAMPLEPREP_SUMMARY            	a C18 colum.n Lipid containing phases were dried under nitrogen and stored at
SP:SAMPLEPREP_SUMMARY            	-80C until they were resuspended for the appropriate analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	All metabolites abundances were calculated from C18 analysis.
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	Relative Abundance
MS_METABOLITE_DATA_START
Samples	A549_Gluc_1	A549_Gluc_2	A549_Gluc_3	A549_Ser_1	A549_Ser_2	A549_Ser_3	H1299_Gluc_1	H1299_Gluc_2	H1299_Gluc_3	H1299_Ser_1	H1299_Ser_2	H1299_Ser_3
Factors	Sample source:A549 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C6] glucose	Sample source:A549 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C6] glucose	Sample source:A549 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C6] glucose	Sample source:A549 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C3] serine	Sample source:A549 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C3] serine	Sample source:A549 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C3] serine	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C6] glucose	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C6] glucose	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C6] glucose	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C3] serine	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C3] serine	Sample source:H1299 cell line | Genotype:Wild Type | Treatment:10pc FBS and [U-13C3] serine
SPB 18:0;O2	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ
SPB 18:1;O2	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ
Cer 18:0;O2/24:0	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ
Cer 18:1;O2/24:0	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ
SM 42:1;O2	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ	NQ
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Retention time	m/z ratio
SPB 18:0;O2	7.24	302.3051
SPB 18:1;O2	7.03	300.2895
Cer 18:0;O2/24:0	16.90	652.6604
Cer 18:1;O2/24:0	16.50	650.6450
SM 42:1;O2	15.59	815.7006
METABOLITES_END
#END