#METABOLOMICS WORKBENCH kwessendorf_20251204_103215 DATATRACK_ID:6839 STUDY_ID:ST004448 ANALYSIS_ID:AN007451 PROJECT_ID:PR002810 VERSION 1 CREATED_ON December 4, 2025, 11:01 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 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 effect to lipid homeostasis of adding back functional LKB1 to an ST:STUDY_TITLE A549 cell line ST:STUDY_SUMMARY Two A549 cell lines, one expressing an empty vector and one expressing ST:STUDY_SUMMARY functional LKB1, were cultured in 10% FBS for 24 hours in high glucose DMEM ST:STUDY_SUMMARY media containing either 13C glucose or 13C serine. Tracing experiments were ST:STUDY_SUMMARY performed in parallel wells to limit variability due to plating or passage ST:STUDY_SUMMARY number accumulation. At the end of the tracing period, wells were washed twice ST:STUDY_SUMMARY with 0.9% w/V saline, then scraped into 100% methanol. Internal standards were ST:STUDY_SUMMARY added and samples were split for polar or neutral lipid analysis by liquid ST:STUDY_SUMMARY chromatography coupled to high-resolution mass spectrometry. MS data was ST:STUDY_SUMMARY analyzed using EL-Maven v0.12.1 with a mass error of 5ppm and maximum retention ST:STUDY_SUMMARY time shift between parent ions and isotopomer ions of 5 seconds. An in-house ST:STUDY_SUMMARY 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 A549 cells expressing an empty vector (+EV) or functional LKB1 (+LKB1) #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_EV_G1 A549_EV_Glucose_1 Sample source:A549 cell line | Genotype:+empty vector | Treatment:10pc FBS and [U-13C6] glucose Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_GLUC_EV_1.mzXML SUBJECT_SAMPLE_FACTORS A549_EV_G2 A549_EV_Glucose_2 Sample source:A549 cell line | Genotype:+empty vector | Treatment:10pc FBS and [U-13C6] glucose Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_GLUC_EV_2.mzXML SUBJECT_SAMPLE_FACTORS A549_EV_G3 A549_EV_Glucose_3 Sample source:A549 cell line | Genotype:+empty vector | Treatment:10pc FBS and [U-13C6] glucose Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_GLUC_EV_3.mzXML SUBJECT_SAMPLE_FACTORS A549_EV_S1 A549_EV_Serine_1 Sample source:A549 cell line | Genotype:+empty vector | Treatment:10pc FBS and [U-13C3] serine Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_Ser_EV_1.mzXML SUBJECT_SAMPLE_FACTORS A549_EV_S2 A549_EV_Serine_2 Sample source:A549 cell line | Genotype:+empty vector | Treatment:10pc FBS and [U-13C3] serine Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_Ser_EV_2.mzXML SUBJECT_SAMPLE_FACTORS A549_EV_S3 A549_EV_Serine_3 Sample source:A549 cell line | Genotype:+empty vector | Treatment:10pc FBS and [U-13C3] serine Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_Ser_EV_3.mzXML SUBJECT_SAMPLE_FACTORS A549_LKB1_G1 A549_LKB1_Glucose_1 Sample source:A549 cell line | Genotype:+LKB1 | Treatment:10pc FBS and [U-13C6] glucose Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_GLUC_LK1_1.mzXML SUBJECT_SAMPLE_FACTORS A549_LKB1_G2 A549_LKB1_Glucose_2 Sample source:A549 cell line | Genotype:+LKB1 | Treatment:10pc FBS and [U-13C6] glucose Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_GLUC_LK1_2.mzXML SUBJECT_SAMPLE_FACTORS A549_LKB1_G3 A549_LKB1_Glucose_3 Sample source:A549 cell line | Genotype:+LKB1 | Treatment:10pc FBS and [U-13C6] glucose Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_GLUC_LK1_3.mzXML SUBJECT_SAMPLE_FACTORS A549_LKB1_S1 A549_LKB1_Serine_1 Sample source:A549 cell line | Genotype:+LKB1 | Treatment:10pc FBS and [U-13C3] serine Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_Ser_LK1_1.mzXML SUBJECT_SAMPLE_FACTORS A549_LKB1_S2 A549_LKB1_Serine_2 Sample source:A549 cell line | Genotype:+LKB1 | Treatment:10pc FBS and [U-13C3] serine Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_Ser_LK1_2.mzXML SUBJECT_SAMPLE_FACTORS A549_LKB1_S3 A549_LKB1_Serine_3 Sample source:A549 cell line | Genotype:+LKB1 | Treatment:10pc FBS and [U-13C3] serine Batch=B2; RAW_FILE_NAME(Raw_File_Name)=A549_Ser_LK1_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. Samples were split in two eppendorfs: one portion was used for CO:COLLECTION_SUMMARY polar lipid analysis on LCMS and the other further extracted and separated to CO:COLLECTION_SUMMARY generate an aqueous layer containing polar metabolites for GCMS analysis and a CO:COLLECTION_SUMMARY hydrophobic phase used for analysis of neutral lipids on LCMS. Separation of CO:COLLECTION_SUMMARY aqueous and organic phases was performed by addition of 200uL of water and 400 CO:COLLECTION_SUMMARY μL of chloroform to the 200 μL of methanol left in the Eppendorf tube allotted CO:COLLECTION_SUMMARY for neutral lipid extraction and analysis on C30. Samples were then vortex and CO:COLLECTION_SUMMARY centrifuged at 21,000gs for 5 min at 4°C. The organic phase was collected and 2 CO:COLLECTION_SUMMARY μL of formic acid was added to the remaining polar phase which was re-extracted CO:COLLECTION_SUMMARY with 400 μL of chloroform. Combined organic phases were dried under nitrogen CO:COLLECTION_SUMMARY and used for neutral lipid analysis. 250 μL of the upper aqueous phase were CO:COLLECTION_SUMMARY transferred to a GC vial and dried at 4°C overnight. 250uL of lower organic CO:COLLECTION_SUMMARY layer was collected and evaporated under nitrogen at room temperature. The CO:COLLECTION_SUMMARY Eppendorfs with 100% methanol, used for polar lipid analysis on C18, were 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 Two A549 cell lines, one expressing an empty vector and one expressing TR:TREATMENT_SUMMARY functional LKB1, were cultured in 10% FBS for 24 hours in high glucose DMEM TR:TREATMENT_SUMMARY media containing either 13C glucose or 13C serine. The goal was to measure the TR:TREATMENT_SUMMARY isotope incorporation into membrane lipids and triglycerides in order to perform TR:TREATMENT_SUMMARY metabolic flux analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Lipids were extracted from cell pellets using 100% methanol for polar lipids on SP:SAMPLEPREP_SUMMARY a C18 column and a modified Folch extraction using a ratio of 6:4:10 of SP:SAMPLEPREP_SUMMARY methanol, water and chloroform. The upper phase was used for metabolomic SP:SAMPLEPREP_SUMMARY analysis on GCMS and the bottom phase was used to measure neutral lipids on a SP:SAMPLEPREP_SUMMARY C30 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 Relative Abundance MS_METABOLITE_DATA_START Samples A549_EV_Glucose_1 A549_EV_Glucose_2 A549_EV_Glucose_3 A549_EV_Serine_1 A549_EV_Serine_2 A549_EV_Serine_3 A549_LKB1_Glucose_1 A549_LKB1_Glucose_2 A549_LKB1_Glucose_3 A549_LKB1_Serine_1 A549_LKB1_Serine_2 A549_LKB1_Serine_3 Factors Sample source:A549 cell line | Genotype:+empty vector | Treatment:10pc FBS and [U-13C6] glucose Sample source:A549 cell line | Genotype:+empty vector | Treatment:10pc FBS and [U-13C6] glucose Sample source:A549 cell line | Genotype:+empty vector | Treatment:10pc FBS and [U-13C6] glucose Sample source:A549 cell line | Genotype:+empty vector | Treatment:10pc FBS and [U-13C3] serine Sample source:A549 cell line | Genotype:+empty vector | Treatment:10pc FBS and [U-13C3] serine Sample source:A549 cell line | Genotype:+empty vector | Treatment:10pc FBS and [U-13C3] serine Sample source:A549 cell line | Genotype:+LKB1 | Treatment:10pc FBS and [U-13C6] glucose Sample source:A549 cell line | Genotype:+LKB1 | Treatment:10pc FBS and [U-13C6] glucose Sample source:A549 cell line | Genotype:+LKB1 | Treatment:10pc FBS and [U-13C6] glucose Sample source:A549 cell line | Genotype:+LKB1 | Treatment:10pc FBS and [U-13C3] serine Sample source:A549 cell line | Genotype:+LKB1 | Treatment:10pc FBS and [U-13C3] serine Sample source:A549 cell line | Genotype:+LKB1 | Treatment:10pc FBS and [U-13C3] serine SPB 18:0;O2 ND ND ND 0.0024 0.00239 0.00237 ND ND ND 0.00244 0.00222 0.0018 SPB 18:1;O2 ND ND ND 0.0101 0.0099 0.00872 ND ND ND 0.0115 0.0126 0.011 Cer 18:0;O2/24:0 ND ND ND 0.031108493 0.043950303 0.035449774 ND ND ND 0.02412469 0.027794754 0.023753551 Cer 18:1;O2/24:0 ND ND ND 0.116883461 0.155084892 0.138483697 ND ND ND 0.121675025 0.13473561 0.12170302 SM 42:1;O2 ND ND ND 0.039282793 0.061299308 0.046105398 ND ND ND 0.02264249 0.038309991 0.038038531 DG 16:0_18:1 ND ND ND 0.104307951 0.15452923 0.090364206 ND ND ND 0.09661867 0.100972847 0.089825282 DG 18:0_18:1 ND ND ND 0.05125723 0.068681839 0.052090861 ND ND ND 0.03431056 0.037705141 0.043841547 PC 34:1 ND ND ND 15.89304084 12.91850872 17.69979206 ND ND ND 21.974856 22.1126219 22.58490902 PC 36:1 ND ND ND 4.216703142 4.245514314 4.89654863 ND ND ND 5.44114046 5.533823802 4.991202474 PE 34:1 ND ND ND 0.959913671 1.496751457 1.745976615 ND ND ND 1.21830876 1.504384926 1.661992589 PE 36:1 ND ND ND 1.737612458 2.193986245 2.302380944 ND ND ND 2.440421795 2.233777546 2.044290235 TG 16:0_18:1_18:0 ND ND ND 0.366195915 0.356712756 0.329013358 ND ND ND 0.299470659 0.275249419 0.31328313 GM3 18:1;O2/16:0 ND ND ND 0.003990267 0.005394018 0.005646169 ND ND ND 0.005824794 0.006373185 0.005679036 GM3 18:1;O2/24:0 ND ND ND 0.014898594 0.017288866 0.01903991 ND ND ND 0.025645308 0.021974813 0.022527774 GM2 18:1;O2/16:0 ND ND ND 0.003986571 0.005082332 0.005535596 ND ND ND 0.005573287 0.006217391 0.005157115 GM2 18:1;O2/24:0 ND ND ND 0.029538349 0.032493363 0.043606126 ND ND ND 0.04584945 0.045605936 0.034410628 GB3 18:1;O2/16:0 ND ND ND 0.000883051 0.001294624 0.001335243 ND ND ND 0.024557388 0.02342901 0.023353253 GB3 18:1;O2/24:0 ND ND ND 0.001162398 0.001665347 0.001394631 ND ND ND 0.043927451 0.041211225 0.037126911 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention time m/z ratio SPB 18:0;O2 6.78 302.304352 SPB 18:1;O2 6.575 300.288788 Cer 18:0;O2/24:0 15.605 652.659607 Cer 18:1;O2/24:0 15.209 650.643311 SM 42:1;O2 14.183 815.699158 DG 16:0_18:1 13.586 612.554993 DG 18:0_18:1 14.535 640.586975 PC 34:1 12.024 760.58313 PC 36:1 12.76 788.614807 PE 34:1 12.016 718.536377 PE 36:1 12.691 746.567871 TG 16:0_18:1_18:0 24.911 878.818359 GM3 18:1;O2/16:0 9.66 1153.7204 GM3 18:1;O2/24:0 11.79 1265.8457 GM2 18:1;O2/16:0 9.65 1356.7998 GM2 18:1;O2/24:0 11.75 1468.9250 GB3 18:1;O2/16:0 10.74 1024.6778 GB3 18:1;O2/24:0 13.16 1136.8031 METABOLITES_END #END