#METABOLOMICS WORKBENCH leaptrkl_20250430_092701 DATATRACK_ID:5873 STUDY_ID:ST003903 ANALYSIS_ID:AN006408 PROJECT_ID:PR002443 VERSION 1 CREATED_ON May 6, 2025, 1:19 pm #PROJECT PR:PROJECT_TITLE Hypertonic stress promotes intracellular lipid accumulation via glutamine PR:PROJECT_TITLE anaplerosis PR:PROJECT_TYPE Untargeted lipidomics PR:PROJECT_SUMMARY In this work, we describe that acute hypertonic stress triggers the upregulation PR:PROJECT_SUMMARY of metabolic pathways that promote glutamine-dependent lipid accumulation PR:PROJECT_SUMMARY associated with decreased oxygen consumption thus increasing potential water PR:PROJECT_SUMMARY storage. PR:INSTITUTE Vanderbilt University PR:DEPARTMENT Chemistry PR:LABORATORY Center for Innovative Technology PR:LAST_NAME Leaptrot PR:FIRST_NAME Katrina PR:ADDRESS 7300 Stevenson Center Lane PR:EMAIL katrina.l.leaptrot@vanderbilt.edu PR:PHONE 6158228422 #STUDY ST:STUDY_TITLE Hypertonic stress promotes intracellular lipid accumulation via glutamine ST:STUDY_TITLE anaplerosis ST:STUDY_TYPE Untargeted lipidomics ST:STUDY_SUMMARY In this work, we describe that acute hypertonic stress triggers the upregulation ST:STUDY_SUMMARY of metabolic pathways that promote glutamine-dependent lipid accumulation ST:STUDY_SUMMARY associated with decreased oxygen consumption thus increasing potential water ST:STUDY_SUMMARY storage. ST:INSTITUTE Vanderbilt University ST:DEPARTMENT Chemistry ST:LABORATORY Center for Innovative Technology ST:LAST_NAME Leaptrot ST:FIRST_NAME Katrina ST:ADDRESS 7300 Stevenson Center Lane ST:EMAIL katrina.l.leaptrot@vanderbilt.edu ST:PHONE 6158228422 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 9 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:CELL_STRAIN_DETAILS kidney inner medullary collecting duct cells #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 - Ctrl 3_neg Sample source:Kidney cells | Treatment:Control RAW_FILE_NAME(MS Sample Name)=005_neg_FS_Ctrl 3_r1_20240604_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - Ctrl 1_neg Sample source:Kidney cells | Treatment:Control RAW_FILE_NAME(MS Sample Name)=006_neg_FS_Ctrl 1_r1_20240604_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - Ctrl 2_neg Sample source:Kidney cells | Treatment:Control RAW_FILE_NAME(MS Sample Name)=013_neg_FS_Ctrl 2_r1_20240604_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - Ctrl 4_neg Sample source:Kidney cells | Treatment:Control RAW_FILE_NAME(MS Sample Name)=014_neg_FS_Ctrl 4_r1_20240604_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - NaCl 4_neg Sample source:Kidney cells | Treatment:NaCl RAW_FILE_NAME(MS Sample Name)=008_neg_FS_NaCl 4_r1_20240604_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - NaCl 1_neg Sample source:Kidney cells | Treatment:NaCl RAW_FILE_NAME(MS Sample Name)=011_neg_FS_NaCl 1_r1_20240604_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - NaCl 3_neg Sample source:Kidney cells | Treatment:NaCl RAW_FILE_NAME(MS Sample Name)=015_neg_FS_NaCl 3_r2_20240604_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - NaCl 5_neg Sample source:Kidney cells | Treatment:NaCl RAW_FILE_NAME(MS Sample Name)=021_neg_FS_NaCl 5_r2_20240604_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - NaCl 2_neg Sample source:Kidney cells | Treatment:NaCl RAW_FILE_NAME(MS Sample Name)=009_neg_FS_NaCl 2_r1_20240604_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - Ctrl 2_pos Sample source:Kidney cells | Treatment:Control RAW_FILE_NAME(MS Sample Name)=028_pos_FS_Ctrl 2_r1_20240521_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - Ctrl 3_pos Sample source:Kidney cells | Treatment:Control RAW_FILE_NAME(MS Sample Name)=031_pos_FS_Ctrl 3_r1_20240521_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - Ctrl 1_pos Sample source:Kidney cells | Treatment:Control RAW_FILE_NAME(MS Sample Name)=033_pos_FS_Ctrl 1_r1_20240521_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - Ctrl 4_pos Sample source:Kidney cells | Treatment:Control RAW_FILE_NAME(MS Sample Name)=036_pos_FS_Ctrl 4_r1_20240521_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - NaCl 5_pos Sample source:Kidney cells | Treatment:NaCl RAW_FILE_NAME(MS Sample Name)=027_pos_FS_NaCl 5_r1_20240521_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - NaCl 3_pos Sample source:Kidney cells | Treatment:NaCl RAW_FILE_NAME(MS Sample Name)=029_pos_FS_NaCl 3_r1_20240521_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - NaCl 4_pos Sample source:Kidney cells | Treatment:NaCl RAW_FILE_NAME(MS Sample Name)=030_pos_FS_NaCl 4_r1_20240521_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - NaCl 1_pos Sample source:Kidney cells | Treatment:NaCl RAW_FILE_NAME(MS Sample Name)=034_pos_FS_NaCl 1_r1_20240521_Arroyo_KLL SUBJECT_SAMPLE_FACTORS - NaCl 2_pos Sample source:Kidney cells | Treatment:NaCl RAW_FILE_NAME(MS Sample Name)=035_pos_FS_NaCl 2_r1_20240521_Arroyo_KLL #COLLECTION CO:COLLECTION_SUMMARY Inner medullary collecting duct cells were cultured in standard low glucose DMEM CO:COLLECTION_SUMMARY supplemented with 10% FBS, antibiotic and antimycotic. Cells were plated in 10 CO:COLLECTION_SUMMARY cm plates and grown to 90% confluency. Medium was the changed to DMEM low CO:COLLECTION_SUMMARY glucose with or without 100 mmol NaCl added. Cells were incubated overnight and CO:COLLECTION_SUMMARY collected for downstream analysis. CO:SAMPLE_TYPE Kidney #TREATMENT TR:TREATMENT_SUMMARY Treatment was DMEM low glucose with or without 100 mmol NaCl. No other TR:TREATMENT_SUMMARY additives. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen samples were thawed on ice and lysed in ice-cold lysis buffer (1:1:2, SP:SAMPLEPREP_SUMMARY acetonitrile: methanol: ammonium bicarbonate 0.1M, pH 8.0) to an equal cell SP:SAMPLEPREP_SUMMARY density per sample, followed by probe tip sonication with 10 pulses at 30% SP:SAMPLEPREP_SUMMARY power. Samples were normalized by total protein amount (150 µg) based on SP:SAMPLEPREP_SUMMARY bicinchoninic acid (BCA) assay and further mixed with 800 µL of cold MeOH, SP:SAMPLEPREP_SUMMARY vortexed for 30 seconds and incubated overnight at -80°C for protein SP:SAMPLEPREP_SUMMARY precipitation. Following incubation, samples were centrifuged for 10 min at SP:SAMPLEPREP_SUMMARY 15,000 rpm at 4°C and the supernatant was transferred to a new labeled tube and SP:SAMPLEPREP_SUMMARY dried down using a cold vacuum centrifuge. Samples were reconstituted in 100 µL SP:SAMPLEPREP_SUMMARY H2O, 100 µL MeOH, and 10 µL of SPLASH LIPIDOMIX with vortex mixing after each SP:SAMPLEPREP_SUMMARY addition. Samples were incubated at room temperature for 10 min followed by SP:SAMPLEPREP_SUMMARY liquid-liquid extraction. For liquid-liquid extraction (LLE), 600 µL MTBE was SP:SAMPLEPREP_SUMMARY added with vortex mixing for 30 seconds followed by incubation on ice for 10 min SP:SAMPLEPREP_SUMMARY and centrifugation at 15,000 rpm for 15 minutes at 4°C. An upper (hydrophobic) SP:SAMPLEPREP_SUMMARY fraction was transferred and dried down using cold vacuum centrifuge and stored SP:SAMPLEPREP_SUMMARY at -80°C for further lipidomic studies. Prior to mass spectrometry analysis, SP:SAMPLEPREP_SUMMARY individual hydrophobic extracts were reconstituted in 80 µL methanol:chloroform SP:SAMPLEPREP_SUMMARY (9:1, v:v) containing exogenous lipid standards to assess instrument SP:SAMPLEPREP_SUMMARY variability. A pooled quality control (QC) sample was prepared by pooling equal SP:SAMPLEPREP_SUMMARY volumes (30 μL) from each individual sample following reconstitution. SP:SAMPLEPREP_PROTOCOL_FILENAME Global_Untargeted_Lipidomics_Methods.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Thermo Hypersil GOLD aQ (100 x 2.1mm,1.9um) CH:SOLVENT_A 100% water; 10 mM ammonium acetate; 0.1% formic acid CH:SOLVENT_B 60% acetonitrile/36% isopropanol/4% water; 10 mM ammonium acetate; 0.1% formic CH:SOLVENT_B acid CH:FLOW_GRADIENT 0.00 min 70% B; 0.50 min 30% B; 2.00 min 70% B; 15.00 min 100% B; 21.00 min 100% CH:FLOW_GRADIENT B; 22.00 min 10% B; 24.00 min 10% B; 25.00 min 30% B; 30 min 70% B CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 40 CH:METHODS_FILENAME Global_Untargeted_Lipidomics_Methods.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE Global_Untargeted_Lipidomics_Methods.pdf #MS MS:INSTRUMENT_NAME Agilent 6560 Ion Mobility MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Mass spectrometry raw data was imported, processed, and normalized to all MS:MS_COMMENTS compounds using Progenesis QI v.3.0 (Non-linear Dynamics, Newcastle, UK). All MS MS:MS_COMMENTS and MS/MS sample runs were aligned against a pooled QC reference run created MS:MS_COMMENTS from equal portions of all experimental samples. QA/QC metrics were assessed MS:MS_COMMENTS throughout the experiment by monitoring retention times and peak areas of SPLASH MS:MS_COMMENTS LIPIDOMIX standards. Relative abundances for both positive and negative ion mode MS:MS_COMMENTS data acquisitions were less than 14% variability for 90% of standards for sample MS:MS_COMMENTS preparation and less than 5% variability for 100% of standards for instrument MS:MS_COMMENTS variability. Annotations (https://doi.org/10.1007/s13361-016-1469-y) were MS:MS_COMMENTS assigned using accurate mass measurements (<10 ppm), isotope distribution MS:MS_COMMENTS similarity scores, and assessment of MS2 spectrum matching (when applicable) MS:MS_COMMENTS from MS Dial database searching, as well as MS2 searches to Lipid Annotator MS:MS_COMMENTS (https://doi.org/10.3390/metabo10030101) and Lipid Match MS:MS_COMMENTS (https://doi.org/10.1186/s12859-017-1744-3). Lipid species significance was MS:MS_COMMENTS assessed by ANOVA (analysis of variance) from normalized compound abundance MS:MS_COMMENTS data. Visualizations of lipidomics annotations and statistics were prepared MS:MS_COMMENTS using Cytoscape 3.10.3 (https://doi.org/10.1101/gr.1239303). MS:MS_RESULTS_FILE ST003903_AN006408_Results.txt UNITS:Abundance Has m/z:Yes Has RT:Yes RT units:Minutes #END