#METABOLOMICS WORKBENCH mkolar_20240920_120313 DATATRACK_ID:5212 STUDY_ID:ST003502 ANALYSIS_ID:AN005750 PROJECT_ID:PR002148 VERSION 1 CREATED_ON September 23, 2024, 2:15 pm #PROJECT PR:PROJECT_TITLE Quantifying acyl-chain diversity in isobaric compound lipids containing PR:PROJECT_TITLE monomethyl branched-chain fatty acids PR:PROJECT_SUMMARY To evaluate whether branched-chain fatty acids (BCFAs) could be effectively PR:PROJECT_SUMMARY separated and quantified using a C30 liquid chromatography column, we treated PR:PROJECT_SUMMARY 3T3-L1 cells with various lipid substrates, including odd-chain, even-chain, and PR:PROJECT_SUMMARY branched-chain fatty acids. We then targeted the incorporation of these lipids PR:PROJECT_SUMMARY into compound lipids and aimed to determine if these isobaric compounds could be PR:PROJECT_SUMMARY separated. Additionally, we engineered 3T3-L1 cells lacking branched-keto acid PR:PROJECT_SUMMARY dehydrogenase A (Bckdha) and validated our approach using a deuterated BCFA. PR:PROJECT_SUMMARY Compound lipids were analyzed via UHPLC-MS, demonstrating that the C30 column PR:PROJECT_SUMMARY successfully separated lipid species containing monomethyl BCFAs from their PR:PROJECT_SUMMARY straight-chain counterparts. PR:INSTITUTE Salk Institute PR:LAST_NAME Kolar PR:FIRST_NAME Matthew PR:ADDRESS 10010 N Torrey Pines Rd, La Jolla, CA, 92037, USA PR:EMAIL mkolar@salk.edu PR:PHONE 8584534100 #STUDY ST:STUDY_TITLE Quantifying acyl-chain diversity in isobaric compound lipids containing ST:STUDY_TITLE monomethyl branched-chain fatty acids ST:STUDY_SUMMARY Compound lipids comprise a diverse group of metabolites present in living ST:STUDY_SUMMARY systems, and metabolic- and environmentally-driven structural distinctions ST:STUDY_SUMMARY across this family is increasingly linked to biological function. However, ST:STUDY_SUMMARY methods for deconvoluting these often isobaric lipid species are lacking or ST:STUDY_SUMMARY require specialized instrumentation. Notably, acyl-chain diversity within cells ST:STUDY_SUMMARY may be influenced by nutritional states, metabolic dysregulation, or genetic ST:STUDY_SUMMARY alterations. Therefore, a reliable, validated method of quantifying structurally ST:STUDY_SUMMARY similar even-, odd-, and branched-chain acyl groups within intact compound ST:STUDY_SUMMARY lipids will be invaluable for gaining molecular insights into their biological ST:STUDY_SUMMARY functions. Here we demonstrate the chromatographic resolution of isobaric lipids ST:STUDY_SUMMARY containing distinct combinations of straight-chain and branched-chain acyl ST:STUDY_SUMMARY groups via ultra-high-pressure liquid chromatography (UHPLC)-mass spectrometry ST:STUDY_SUMMARY (MS) using a C30 liquid chromatography column. Using metabolically-engineered ST:STUDY_SUMMARY adipocytes lacking branched-keto acid dehydrogenase A (Bckdha), we validate this ST:STUDY_SUMMARY approach through a combination of fatty acid supplementation and metabolic ST:STUDY_SUMMARY tracing using monomethyl branched-chain fatty acids and valine. We observe ST:STUDY_SUMMARY resolution of numerous isobaric triacylglycerols and other compound lipids, ST:STUDY_SUMMARY demonstrating the resolving utility of this method. This approach strengthens ST:STUDY_SUMMARY the ability to quantify and characterize the inherent diversity of acyl chains ST:STUDY_SUMMARY across the lipidome. ST:INSTITUTE Salk Institute for Biological Studies ST:DEPARTMENT Molecular and Cell Biology ST:LABORATORY Metallo Lab ST:LAST_NAME Kolar ST:FIRST_NAME Matthew ST:ADDRESS 10010 N Torrey Pines Rd ST:EMAIL mkolar@salk.edu ST:PHONE 2524534100 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:CELL_STRAIN_DETAILS 3T3-L1 #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 - 3T3L1_OCFA_1 Sample source:3T3-L1 cells | Genotype:WT | Treatment:FA 15 0 FA 17 0 RAW_FILE_NAME(Raw file name)=3T3L1_ControlKO_OCFA_noB12_C30_1.mzML SUBJECT_SAMPLE_FACTORS - 3T3L1_OCFA_2 Sample source:3T3-L1 cells | Genotype:WT | Treatment:FA 15 0 FA 17 0 RAW_FILE_NAME(Raw file name)=3T3L1_ControlKO_OCFA_noB12_C30_2.mzML SUBJECT_SAMPLE_FACTORS - 3T3L1_OCFA_3 Sample source:3T3-L1 cells | Genotype:WT | Treatment:FA 15 0 FA 17 0 RAW_FILE_NAME(Raw file name)=3T3L1_ControlKO_OCFA_noB12_C30_3.mzML SUBJECT_SAMPLE_FACTORS - 3T3L1_BCFA_1 Sample source:3T3-L1 cells | Genotype:WT | Treatment:iso-FA 16 0 Iso-FA 17 0 anteiso-FA 17 0 RAW_FILE_NAME(Raw file name)=3T3L1_ControlKO_BCFA_noB12_C30_1.mzML SUBJECT_SAMPLE_FACTORS - 3T3L1_BCFA_2 Sample source:3T3-L1 cells | Genotype:WT | Treatment:iso-FA 16 0 Iso-FA 17 0 anteiso-FA 17 0 RAW_FILE_NAME(Raw file name)=3T3L1_ControlKO_BCFA_noB12_C30_2.mzML SUBJECT_SAMPLE_FACTORS - 3T3L1_BCFA_3 Sample source:3T3-L1 cells | Genotype:WT | Treatment:iso-FA 16 0 Iso-FA 17 0 anteiso-FA 17 0 RAW_FILE_NAME(Raw file name)=3T3L1_ControlKO_BCFA_noB12_C30_3.mzML SUBJECT_SAMPLE_FACTORS - 3T3L1_ECFA_1 Sample source:3T3-L1 cells | Genotype:WT | Treatment:FA 16 0 FA16 1(9Z) RAW_FILE_NAME(Raw file name)=3T3L1_ControlKO_ECFA_noB12_C30_1.mzML SUBJECT_SAMPLE_FACTORS - 3T3L1_ECFA_2 Sample source:3T3-L1 cells | Genotype:WT | Treatment:FA 16 0 FA16 1(9Z) RAW_FILE_NAME(Raw file name)=3T3L1_ControlKO_ECFA_noB12_C30_2.mzML SUBJECT_SAMPLE_FACTORS - 3T3L1_ECFA_3 Sample source:3T3-L1 cells | Genotype:WT | Treatment:FA 16 0 FA16 1(9Z) RAW_FILE_NAME(Raw file name)=3T3L1_ControlKO_ECFA_noB12_C30_3.mzML SUBJECT_SAMPLE_FACTORS - 3T3L1_2HBCFA_BckdhaKO_1 Sample source:3T3-L1 cells | Genotype:BckdhaKO | Treatment:iso-FA 16 0[D7] RAW_FILE_NAME(Raw file name)=3T3L1_BckdhaKO_2HBCFA_withB12_C30_1.mzML SUBJECT_SAMPLE_FACTORS - 3T3L1_2HBCFA_BckdhaKO_2 Sample source:3T3-L1 cells | Genotype:BckdhaKO | Treatment:iso-FA 16 0[D7] RAW_FILE_NAME(Raw file name)=3T3L1_BckdhaKO_2HBCFA_withB12_C30_2.mzML SUBJECT_SAMPLE_FACTORS - 3T3L1_2HBCFA_BckdhaKO_3 Sample source:3T3-L1 cells | Genotype:BckdhaKO | Treatment:iso-FA 16 0[D7] RAW_FILE_NAME(Raw file name)=3T3L1_BckdhaKO_2HBCFA_withB12_C30_3.mzML #COLLECTION CO:COLLECTION_SUMMARY Cells were washed with cold PBS and afterwards subjected to lipid extraction. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY WT 3T3-L1 cells were treated with 100 uM of the respective fatty acid mixture of TR:TREATMENT_SUMMARY ECFAs vs BCFAs or OCFAs for 96 hours prior to lipid extraction. The bckdhaKO TR:TREATMENT_SUMMARY 3T3-L1 cells were treated with 100 uM of iso-FA 16:0[D7]-BSA complex for 96 TR:TREATMENT_SUMMARY hours prior to lipid extraction. These cells with the iso-FA 16:0[D7] were also TR:TREATMENT_SUMMARY treated with cobalamin 500 nM. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Lipid extraction was carried out using a modified Folch SP:SAMPLEPREP_SUMMARY methanol/chloroform/water extraction at a ratio of 5:5:2 with the inclusion of SP:SAMPLEPREP_SUMMARY 10nmol FA 12:0 dodecylglycerol, 10nmol palmitate[D31], 10ng of each lipid in the SP:SAMPLEPREP_SUMMARY Avanti EquiSPLASH mix. The methanol phase was washed a second time with SP:SAMPLEPREP_SUMMARY chloroform after addition of 2μL formic acid. The chloroform phase (bottom SP:SAMPLEPREP_SUMMARY layer) was transferred and dried under nitrogen gas at room temperature. Samples SP:SAMPLEPREP_SUMMARY were stored at -20 °C before analysis by LC-MS/MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The LC gradient ran from 30% to 43% B from 3-8 min, then from 43% to 50% B from CH:CHROMATOGRAPHY_SUMMARY 8-9 min, then 50 to 90% B from 9-18 min, then 90 to 99% B from 18-26 min, then CH:CHROMATOGRAPHY_SUMMARY held at 99% B from 26-30 min, before returning to 30% B in 6 min and held for a CH:CHROMATOGRAPHY_SUMMARY further 4 min CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Accucore C30 coulmn (2.6 µm, 250 x 2.1 mm) CH:SOLVENT_A 40:60 v/v water:acetonitrile with 10 mM ammonium formate and 0.1% formic acid CH:SOLVENT_B 10:90:0.1 v/v/v acetonitrile:2-propanol:water with 10 mM ammonium formate and CH:SOLVENT_B 0.1% formic acid CH:FLOW_GRADIENT 0 min, 30%B; 3-8 min, 30-43%B; 8-9 min, 43-50%B; 9-18 min, 50-90%B, 18-26 min CH:FLOW_GRADIENT 90-99%B, 26-30 min, 99%B, 30-40 min, 30% B CH:FLOW_RATE 0.2 ml/min CH:COLUMN_TEMPERATURE 40 #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 El-MAVEN #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AUC MS_METABOLITE_DATA_START Samples 3T3L1_OCFA_1 3T3L1_OCFA_2 3T3L1_OCFA_3 3T3L1_BCFA_1 3T3L1_BCFA_2 3T3L1_BCFA_3 3T3L1_ECFA_1 3T3L1_ECFA_2 3T3L1_ECFA_3 3T3L1_2HBCFA_BckdhaKO_1 3T3L1_2HBCFA_BckdhaKO_2 3T3L1_2HBCFA_BckdhaKO_3 Factors Sample source:3T3-L1 cells | Genotype:WT | Treatment:FA 15 0 FA 17 0 Sample source:3T3-L1 cells | Genotype:WT | Treatment:FA 15 0 FA 17 0 Sample source:3T3-L1 cells | Genotype:WT | Treatment:FA 15 0 FA 17 0 Sample source:3T3-L1 cells | Genotype:WT | Treatment:iso-FA 16 0 Iso-FA 17 0 anteiso-FA 17 0 Sample source:3T3-L1 cells | Genotype:WT | Treatment:iso-FA 16 0 Iso-FA 17 0 anteiso-FA 17 0 Sample source:3T3-L1 cells | Genotype:WT | Treatment:iso-FA 16 0 Iso-FA 17 0 anteiso-FA 17 0 Sample source:3T3-L1 cells | Genotype:WT | Treatment:FA 16 0 FA16 1(9Z) Sample source:3T3-L1 cells | Genotype:WT | Treatment:FA 16 0 FA16 1(9Z) Sample source:3T3-L1 cells | Genotype:WT | Treatment:FA 16 0 FA16 1(9Z) Sample source:3T3-L1 cells | Genotype:BckdhaKO | Treatment:iso-FA 16 0[D7] Sample source:3T3-L1 cells | Genotype:BckdhaKO | Treatment:iso-FA 16 0[D7] Sample source:3T3-L1 cells | Genotype:BckdhaKO | Treatment:iso-FA 16 0[D7] TG 47:0 5418430464 5835490304 4005431552 1934597248 4264292608 4583374336 3659693312 5345233920 5528838656 401486432 250523840 353902592 TG 48:0 7707893248 8314024448 5608640000 3919606272 8116362752 8999619584 7362956800 11286762496 11656099840 9553313792 6857595904 8635877376 DG 31:0 7249204 7018597 4147741 1738016 5843641 5579079 3033817 4819800 5900321 376314 276629 342535 TG 48:0[D7] 0 0 0 0 0 0 0 0 0 5124629504 4166782720 4872848384 TG48:0[D14] 0 0 0 0 0 0 0 0 0 3055317760 2894462720 3118973440 TG48:0[D21] 0 0 0 0 0 0 0 0 0 761830336 812348480 827714304 TG 48:1 20071106560 20454535168 14610538496 9324103680 19999113216 21150238720 18853081088 25356490752 26306648064 21459685376 15297381376 19644293120 TG 48:1 [D7] 0 0 0 0 0 0 0 0 0 13576557568 11454684160 13766806528 TG 48:1[D14] 0 0 0 0 0 0 0 0 0 5053838848 4951851520 5191706624 PC 32:0 1223379712 1355554688 731506304 339591200 1222728576 1350010240 819142080 1858336640 2001339776 1581349120 1336824320 1695342080 PC 32:0[D7] 0 0 0 0 0 0 0 0 0 3349406976 3492854016 3812318720 PC 32:0[D14] 0 0 0 0 0 0 0 0 0 766249856 916881472 889947072 PC 34:0 250515792 272292256 142976224 52850848 216793504 219960464 123018392 343170336 372737984 216279824 197321840 225402384 PC 34:0[D7] 0 0 0 0 0 0 0 0 0 273044480 285069664 333329216 PC 34:0[D14] 0 0 0 0 0 0 0 0 0 22418544 26907786 29124420 PC 34:1 11445960704 12132649984 6943326208 2240319232 8024037376 7882993664 5098124288 10071073792 10567833600 6879485440 5222950912 6731740160 PC 34:1[D7] 0 0 0 0 0 0 0 0 0 1933054336 1896840704 2104757888 PC O-32:0 67748584 75943856 37270676 11255790 61403440 61370372 31002566 97629656 114925448 64391304 48355900 71379680 PC O-32:0[D7] 0 0 0 0 0 0 0 0 0 91829168 87102504 107918152 PE 34:0 1518356736 1737688704 917958400 215655264 882414656 921989824 439455968 948769216 1156768896 88993040 58033784 90135976 PE 34:0[D7] 0 0 0 0 0 0 0 0 0 51774800 47683056 53959180 Cer 18:1;O2/16:0 39206276 40562416 29187608 22372126 62318908 49739748 36891148 54282244 63218556 60687944 53600164 60035332 Cer 34:1;O2[D7] 0 0 0 0 0 0 0 0 0 1342818.62 1427882 1254681.38 SM 34:1;O2 1763653632 1922340480 1107199616 637715328 2226674944 2160127232 1078895488 2337356032 2448612864 2146912768 1635727232 2304371200 SM 34:1;O2[D7] 0 0 0 0 0 0 0 0 0 64200856 56627816 68559240 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quanitated m/z RT (min) TG 47:0 810.7545 26.02 TG 48:0 824.7702 26.32 DG 31:0 572.5249 21.21 TG 48:0[D7] 831.8141 26.02 TG48:0[D14] 838.856 25.89 TG48:0[D21] 845.902 25.66 TG 48:1 822.7545 25.53 TG 48:1 [D7] 829.7982 25.32 TG 48:1[D14] 836.8414 25.12 PC 32:0 734.5694 19.97 PC 32:0[D7] 741.6134 19.81 PC 32:0[D14] 748.6573 19.61 PC 34:0 762.6007 20.78 PC 34:0[D7] 769.6447 20.63 PC 34:0[D14] 776.6886 20.44 PC 34:1 760.5851 20.07 PC 34:1[D7] 767.629 19.84 PC O-32:0 720.5902 20.53 PC O-32:0[D7] 727.6341 20.36 PE 34:0 720.5538 19.55 PE 34:0[D7] 727.5977 19.36 Cer 18:1;O2/16:0 538.5194 20.18 Cer 34:1;O2[D7] 545.5633 19.99 SM 34:1;O2 703.5748 19.13 SM 34:1;O2[D7] 710.6188 18.93 METABOLITES_END #END