#METABOLOMICS WORKBENCH basilm_20230203_130930 DATATRACK_ID:3721 STUDY_ID:ST002504 ANALYSIS_ID:AN004120 PROJECT_ID:PR001618 VERSION 1 CREATED_ON March 13, 2023, 12:01 pm #PROJECT PR:PROJECT_TITLE Untargeted lipidomics of C. elegans upon depletion of ash-2 and prx-5. PR:PROJECT_SUMMARY Untargeted lipidomics in middle-aged C. elegans upon enrichment of PR:PROJECT_SUMMARY monounsaturated fatty acids by ash-2 RNAi and peroxisome depletion by prx-5 PR:PROJECT_SUMMARY RNAi. PR:INSTITUTE Stanford University PR:LAST_NAME Papsdorf PR:FIRST_NAME Katharina PR:ADDRESS 290 Jane Stanford way, 94301 Palo Alto, CA, USA PR:EMAIL papsdorf@stanford.edu PR:PHONE +1 650 546 5366 #STUDY ST:STUDY_TITLE Lipid droplets and peroxisomes are co-regulated to drive lifespan extension in ST:STUDY_TITLE response to mono-unsaturated fatty acids ST:STUDY_SUMMARY Dietary mono-unsaturated fatty acids (MUFAs) are linked to human longevity and ST:STUDY_SUMMARY extend lifespan in several species. But the mechanisms by which MUFAs extend ST:STUDY_SUMMARY lifespan remain unclear. Here we show that an organelle network involving lipid ST:STUDY_SUMMARY droplets and peroxisomes is critical for lifespan extension by MUFAs in C. ST:STUDY_SUMMARY elegans. MUFA accumulation increases lipid droplet number in fat storage ST:STUDY_SUMMARY tissues, and this is necessary for MUFA-induced longevity. Lipid droplet number ST:STUDY_SUMMARY in young or middle-aged individuals can predict remaining lifespan, consistent ST:STUDY_SUMMARY with a beneficial effect of lipid droplets on lifespan. Lipidomics datasets ST:STUDY_SUMMARY reveal that MUFA accumulation also modifies the ratio of membrane lipids and ST:STUDY_SUMMARY ether lipids, a signature predictive of decreased lipid oxidation. We validate ST:STUDY_SUMMARY that MUFAs decrease lipid oxidation in middle-aged individuals, and that this is ST:STUDY_SUMMARY important for MUFA-induced longevity. Intriguingly, the increase in lipid ST:STUDY_SUMMARY droplet number in response to MUFAs is accompanied by a concomitant increase in ST:STUDY_SUMMARY peroxisome number. Using a targeted screen, we identify genes involved in the ST:STUDY_SUMMARY co-regulation or uncoupling of this lipid droplet-peroxisome network. We find ST:STUDY_SUMMARY that induction of both organelles is optimal for lifespan extension. Our study ST:STUDY_SUMMARY uncovers an organelle network involved in lipid homeostasis and lifespan ST:STUDY_SUMMARY regulation and identifies a mechanism of action for MUFAs to extend lifespan, ST:STUDY_SUMMARY opening new avenues for lipid-based interventions to delay aging. For the ST:STUDY_SUMMARY manuscript only the conditions “control” and “ash-2 RNAi” are plotted ST:INSTITUTE Stanford University ST:LAST_NAME Papsdorf ST:FIRST_NAME Katharina ST:ADDRESS 290 Jane Stanford way, 94301 Palo Alto, CA, USA ST:EMAIL papsdorf@stanford.edu ST:PHONE +1 650 546 5366 #SUBJECT SU:SUBJECT_TYPE Invertebrate SU:SUBJECT_SPECIES Caenorhabditis elegans SU:TAXONOMY_ID 6239 SU:AGE_OR_AGE_RANGE middle-aged (adult day 5) #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 - ASH10 Genotype:Wild-type | Treatment:ash-2 RNAi RAW_FILE_NAME=ASH10.raw SUBJECT_SAMPLE_FACTORS - ASH14 Genotype:Wild-type | Treatment:ash-2 RNAi RAW_FILE_NAME=ASH14.raw SUBJECT_SAMPLE_FACTORS - ASH18 Genotype:Wild-type | Treatment:ash-2 RNAi RAW_FILE_NAME=ASH18.raw SUBJECT_SAMPLE_FACTORS - ASH2 Genotype:Wild-type | Treatment:ash-2 RNAi RAW_FILE_NAME=ASH2.raw SUBJECT_SAMPLE_FACTORS - ASH22 Genotype:Wild-type | Treatment:ash-2 RNAi RAW_FILE_NAME=ASH22.raw SUBJECT_SAMPLE_FACTORS - ASH6 Genotype:Wild-type | Treatment:ash-2 RNAi RAW_FILE_NAME=ASH6.raw SUBJECT_SAMPLE_FACTORS - EV1 Genotype:Wild-type | Treatment:Control RAW_FILE_NAME=EV1.raw SUBJECT_SAMPLE_FACTORS - EV13 Genotype:Wild-type | Treatment:Control RAW_FILE_NAME=EV13.raw SUBJECT_SAMPLE_FACTORS - EV17 Genotype:Wild-type | Treatment:Control RAW_FILE_NAME=EV17.raw SUBJECT_SAMPLE_FACTORS - EV21 Genotype:Wild-type | Treatment:Control RAW_FILE_NAME=EV21.raw SUBJECT_SAMPLE_FACTORS - EV5 Genotype:Wild-type | Treatment:Control RAW_FILE_NAME=EV5.raw SUBJECT_SAMPLE_FACTORS - EV9 Genotype:Wild-type | Treatment:Control RAW_FILE_NAME=EV9.raw SUBJECT_SAMPLE_FACTORS - P-A11 Genotype:Wild-type | Treatment:ash-2/prx-5 RNAi RAW_FILE_NAME=P-A11.raw SUBJECT_SAMPLE_FACTORS - P-A15 Genotype:Wild-type | Treatment:ash-2/prx-5 RNAi RAW_FILE_NAME=P-A15.raw SUBJECT_SAMPLE_FACTORS - P-A19 Genotype:Wild-type | Treatment:ash-2/prx-5 RNAi RAW_FILE_NAME=P-A19.raw SUBJECT_SAMPLE_FACTORS - P-A23 Genotype:Wild-type | Treatment:ash-2/prx-5 RNAi RAW_FILE_NAME=P-A23.raw SUBJECT_SAMPLE_FACTORS - P-A3 Genotype:Wild-type | Treatment:ash-2/prx-5 RNAi RAW_FILE_NAME=P-A3.raw SUBJECT_SAMPLE_FACTORS - P-A7 Genotype:Wild-type | Treatment:ash-2/prx-5 RNAi RAW_FILE_NAME=P-A7.raw SUBJECT_SAMPLE_FACTORS - PRX12 Genotype:Wild-type | Treatment:prx-5 RNAi RAW_FILE_NAME=PRX12.raw SUBJECT_SAMPLE_FACTORS - PRX16 Genotype:Wild-type | Treatment:prx-5 RNAi RAW_FILE_NAME=PRX16.raw SUBJECT_SAMPLE_FACTORS - PRX20 Genotype:Wild-type | Treatment:prx-5 RNAi RAW_FILE_NAME=PRX20.raw SUBJECT_SAMPLE_FACTORS - PRX24 Genotype:Wild-type | Treatment:prx-5 RNAi RAW_FILE_NAME=PRX24.raw SUBJECT_SAMPLE_FACTORS - PRX4 Genotype:Wild-type | Treatment:prx-5 RNAi RAW_FILE_NAME=PRX4.raw SUBJECT_SAMPLE_FACTORS - PRX8 Genotype:Wild-type | Treatment:prx-5 RNAi RAW_FILE_NAME=PRX8.raw SUBJECT_SAMPLE_FACTORS - QC_MSMS Genotype:- | Treatment:- RAW_FILE_NAME=- SUBJECT_SAMPLE_FACTORS - blank Genotype:- | Treatment:- RAW_FILE_NAME=- #COLLECTION CO:COLLECTION_SUMMARY At middle-age (adult day 5), worms were transferred to empty RNAi plates without CO:COLLECTION_SUMMARY any bacteria for 15 minutes, to clear residual bacteria in the gut. Worms were CO:COLLECTION_SUMMARY then collected in 200 µl M9 in protein-low bind Eppendorf tubes (cat # CO:COLLECTION_SUMMARY 13-698-794). Worms were lysed using a pre-chilled stainless-steel homogenizer CO:COLLECTION_SUMMARY (Wheaton, cat # 357572) and were homogenized with 15 plunger strokes and protein CO:COLLECTION_SUMMARY concentration of the lysate was determined using the Pierce BCA Protein Assay CO:COLLECTION_SUMMARY Kit (Thermo- Scientific). The lysate (from approximately 500 worms) was frozen CO:COLLECTION_SUMMARY on dry ice and stored at -80°C CO:SAMPLE_TYPE Whole worm lysate #TREATMENT TR:TREATMENT_SUMMARY Hermaphrodites were treated with control (empty vector) RNAi, prx-5, ash-2 or TR:TREATMENT_SUMMARY prx-5/ash-2 RNAi until middle-age (adult day 6). Each condition consists of six TR:TREATMENT_SUMMARY biological replicates. To retrieve a large number of age-synchronized worms, TR:TREATMENT_SUMMARY approximately 500 eggs were laid by age-synchronized adult day 1 wild type TR:TREATMENT_SUMMARY parents per replicate plate. After 2 hours of egg laying, the parents were TR:TREATMENT_SUMMARY removed, and the plates were checked that no parents remained. Once the worms TR:TREATMENT_SUMMARY reached the young adult stage, they were washed each day to separate the adult TR:TREATMENT_SUMMARY worms from larvae/eggs. For this, worms were collected in M9 buffer (22 mM TR:TREATMENT_SUMMARY KH2PO4, 34 mM K2HPO4, 86 mM NaCl, 1mM MgSO4) and allowed to settle to the bottom TR:TREATMENT_SUMMARY of the tube. The supernatant was removed and fresh M9 was added. This washing TR:TREATMENT_SUMMARY procedure was repeated 6 times and the adult worms were transferred to fresh TR:TREATMENT_SUMMARY 6-cm RNAi plates seeded with 500 µl RNAi-expressing HT115 bacteria. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Lipids from the whole worm lysates were extracted using a biphasic separation SP:SAMPLEPREP_SUMMARY with methyl tert-butyl ether (MTBE), methanol and water as described previously SP:SAMPLEPREP_SUMMARY (PMID 30532037, PMID 32612231). All reagents used are for lipidomics were LC/MS SP:SAMPLEPREP_SUMMARY grade. Briefly, 298 μl of ice-cold methanol and 2 μl of internal standard SP:SAMPLEPREP_SUMMARY (equiSPLASH, Avanti Polar Lipids, cat# 330731) were added to 50 μl of worm SP:SAMPLEPREP_SUMMARY lysate. The mixture was vortexed for 20 seconds and 1000 μl of ice-cold MTBE SP:SAMPLEPREP_SUMMARY was added. The mixture was incubated under agitation for 30 minutes at 4°C. SP:SAMPLEPREP_SUMMARY After addition of 250 μl of water, the samples were vortexed for 1 minute and SP:SAMPLEPREP_SUMMARY centrifuged at 14,000 g for 10 minutes at room temperature. The upper phase SP:SAMPLEPREP_SUMMARY containing the lipids was collected and dried down under nitrogen. The dry SP:SAMPLEPREP_SUMMARY extracts were reconstituted with 300 μl of 9:1 methanol:toluene (Fisher SP:SAMPLEPREP_SUMMARY Scientific) with 10 mM of ammonium acetate (Sigma Aldrich) and centrifuged at SP:SAMPLEPREP_SUMMARY 14,000 g for 5 minutes before analysis. Water extracted using the same protocol SP:SAMPLEPREP_SUMMARY was used as a blank control. Samples were randomized in all cases during lipid SP:SAMPLEPREP_SUMMARY extraction. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Lipid extracts were analyzed in a randomized order using an Ultimate 3000 RSLC CH:CHROMATOGRAPHY_SUMMARY system coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific) as CH:CHROMATOGRAPHY_SUMMARY previously described (PMID: 32612231). To identify complex lipids, isolated CH:CHROMATOGRAPHY_SUMMARY lipids were analyzed with untargeted lipidomics using liquid chromatography CH:CHROMATOGRAPHY_SUMMARY coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific) (LC/MS). CH:CHROMATOGRAPHY_SUMMARY Lipids were separated using an Accucore C30 column 2.1 x 150 mm, 2.6 μm (Thermo CH:CHROMATOGRAPHY_SUMMARY Scientific, cat# 27826-152130) and mobile phase solvents consisted in 1 mM CH:CHROMATOGRAPHY_SUMMARY ammonium formate and 0.1% formic acid in 60/40 acetonitrile/water (A) and 1 mM CH:CHROMATOGRAPHY_SUMMARY ammonium formate and 0.1% formic acid in 90/10 isopropanol/acetonitrile (B). The CH:CHROMATOGRAPHY_SUMMARY gradient profile used was 30% B for 3 minutes, 30–43% B over 5 minutes, CH:CHROMATOGRAPHY_SUMMARY 43–50% B over 1 minute, 55–90% B over 9 minutes, 90-99% B over 9 minutes and CH:CHROMATOGRAPHY_SUMMARY 99% B for 5 minutes. Lipids were eluted from the column at 0.2 ml/min, the oven CH:CHROMATOGRAPHY_SUMMARY temperature was set at 30°C, and the injection volume was 15 μl. Autosampler CH:CHROMATOGRAPHY_SUMMARY temperature was set at 15°C to prevent lipid aggregation. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME Thermo Accucore C30 (150 x 2.1mm,2.6um) CH:SOLVENT_A 60% acetonitrile/40% water; 1mM ammonium formate; 0.1% formic acid CH:SOLVENT_B 90% isopropanol/10% acetonitrile; 1mM ammonium formate; 0.1% formic acid CH:FLOW_GRADIENT The gradient profile used was 30% B for 3 minutes, 30–43% B over 5 minutes, CH:FLOW_GRADIENT 43–50% B over 1 minute, 55–90% B over 9 minutes, 90-99% B over 9 minutes and CH:FLOW_GRADIENT 99% B for 5 minutes. CH:FLOW_RATE 0.2 ml/min CH:COLUMN_TEMPERATURE 30 CH:SAMPLE_INJECTION 5ul #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 LC-MS peak extraction, alignment, quantification and annotation was performed MS:MS_COMMENTS using LipidSearch software version 4.2.21 (Thermo Fisher Scientific). Lipids MS:MS_COMMENTS were identified by matching the precursor ion mass to a database and the MS:MS_COMMENTS experimental MS/MS spectra to a spectral library containing theoretical MS:MS_COMMENTS fragmentation spectra. To reduce the risk of misidentification, MS/MS spectra MS:MS_COMMENTS from lipids of interest were validated as follows: 1) both positive and negative MS:MS_COMMENTS mode MS/MS spectra match the expected fragments, 2) the main lipid adduct forms MS:MS_COMMENTS detected in positive and negative modes agree with the lipid class identified, MS:MS_COMMENTS 3) the retention time is compatible with the lipid class identified and 4) the MS:MS_COMMENTS peak shape is acceptable. The fragmentation pattern of each lipid class was MS:MS_COMMENTS experimentally validated using lipid internal standards. Single-point internal MS:MS_COMMENTS standard calibrations were used to estimate absolute concentrations using one MS:MS_COMMENTS internal standard for each lipid class. In cases with no exact lipid standard MS:MS_COMMENTS available lipids with molecular similarity were used. Further data processing MS:MS_COMMENTS was done using an in-house analysis pipeline written in R (Version 3.6.3, MS:MS_COMMENTS available in Github at https://github.com/brunetlab). Briefly, processing for MS:MS_COMMENTS samples and spike-in standards were done in the same way. All ions for one lipid MS:MS_COMMENTS were aggregated and lipids with a signal <0 discarded from further analysis. MS:MS_COMMENTS Lipid species were quantified using the corresponding internal standard MS:MS_COMMENTS (equiSPLASH, Avanti Polar Lipids, cat# 330731) for each lipid class. Lipids with MS:MS_COMMENTS signals lower than 3x blank signal were discarded. Lipids with more than 50% of MS:MS_COMMENTS missing values were discarded, and for the remaining missing values, imputation MS:MS_COMMENTS was performed. For this, a value was randomly assigned based on the bottom 5% MS:MS_COMMENTS for the corresponding lipid. Lipids were filtered for a coefficient of variance MS:MS_COMMENTS <0.5. Each sample was divided by its corresponding protein concentration to MS:MS_COMMENTS correct for sample input variations (protein concentrations can be found at MS:MS_COMMENTS https://github.com/brunetlab/Papsdorf_etal_2023). To calculate normalized MS:MS_COMMENTS abundance, each lipid within a sample was divided by the sample median followed MS:MS_COMMENTS by multiplication with the global median. This resulted in a total of 499 MS:MS_COMMENTS filtered and normalized lipids belonging to 16 lipid classes. For a list of MS:MS_COMMENTS identified lipid ions using LipidSearch see MS:MS_COMMENTS https://github.com/brunetlab/Papsdorf_etal_2023. MS:MS_RESULTS_FILE ST002504_AN004120_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END