#METABOLOMICS WORKBENCH Stopka28_20220512_103205_mwtab.txt DATATRACK_ID:3246 STUDY_ID:ST002219 ANALYSIS_ID:AN003628 PROJECT_ID:PR001417 VERSION 1 CREATED_ON July 14, 2022, 3:06 pm #PROJECT PR:PROJECT_TITLE Spatially resolved characterization of tissue metabolic compartments in fasted PR:PROJECT_TITLE and high-fat diet livers PR:PROJECT_SUMMARY Cells adapt their metabolism to physiological stimuli, and metabolic PR:PROJECT_SUMMARY heterogeneity exists between cell types, within tissues, and subcellular PR:PROJECT_SUMMARY compartments. The liver plays an essential role in maintaining whole-body PR:PROJECT_SUMMARY metabolic homeostasis and is structurally defined by metabolic zones. These PR:PROJECT_SUMMARY zones are well-understood on the transcriptomic level, but have not been PR:PROJECT_SUMMARY comprehensively characterized on the metabolomic level. Mass spectrometry PR:PROJECT_SUMMARY imaging (MSI) can be used to map hundreds of metabolites directly from a tissue PR:PROJECT_SUMMARY section, offering an important advance to investigate metabolic heterogeneity in PR:PROJECT_SUMMARY tissues compared to extraction-based metabolomics methods that analyze tissue PR:PROJECT_SUMMARY metabolite profiles in bulk. We established a workflow for the preparation of PR:PROJECT_SUMMARY tissue specimens for matrix-assisted laser desorption/ionization (MALDI) MSI PR:PROJECT_SUMMARY that can be implemented to achieve broad coverage of central carbon, nucleotide, PR:PROJECT_SUMMARY and lipid metabolism pathways. Herein, we used this approach to visualize the PR:PROJECT_SUMMARY effect of nutrient stress and excess on liver metabolism. Our data revealed a PR:PROJECT_SUMMARY highly organized metabolic tissue compartmentalization in livers, which becomes PR:PROJECT_SUMMARY disrupted under high fat diet. Fasting caused changes in the abundance of PR:PROJECT_SUMMARY several metabolites, including increased levels of fatty acids and TCA PR:PROJECT_SUMMARY intermediates while fatty livers had higher levels of purine and pentose PR:PROJECT_SUMMARY phosphate-related metabolites, which generate reducing equivalents to counteract PR:PROJECT_SUMMARY oxidative stress. This spatially conserved approach allowed the visualization of PR:PROJECT_SUMMARY liver metabolic compartmentalization at 30 µm pixel resolution and can be PR:PROJECT_SUMMARY applied more broadly to yield new insights into metabolic heterogeneity in vivo. PR:INSTITUTE Brigham and Women's Hospital PR:LAST_NAME Stopka PR:FIRST_NAME Sylwia PR:ADDRESS 60 Fenway Rd PR:EMAIL sstopka@bwh.harvard.edu PR:PHONE 617-525-9746 #STUDY ST:STUDY_TITLE Spatially resolved characterization of tissue metabolic compartments in fasted ST:STUDY_TITLE and high-fat diet livers ST:STUDY_SUMMARY Cells adapt their metabolism to physiological stimuli, and metabolic ST:STUDY_SUMMARY heterogeneity exists between cell types, within tissues, and subcellular ST:STUDY_SUMMARY compartments. The liver plays an essential role in maintaining whole-body ST:STUDY_SUMMARY metabolic homeostasis and is structurally defined by metabolic zones. These ST:STUDY_SUMMARY zones are well-understood on the transcriptomic level, but have not been ST:STUDY_SUMMARY comprehensively characterized on the metabolomic level. Mass spectrometry ST:STUDY_SUMMARY imaging (MSI) can be used to map hundreds of metabolites directly from a tissue ST:STUDY_SUMMARY section, offering an important advance to investigate metabolic heterogeneity in ST:STUDY_SUMMARY tissues compared to extraction-based metabolomics methods that analyze tissue ST:STUDY_SUMMARY metabolite profiles in bulk. We established a workflow for the preparation of ST:STUDY_SUMMARY tissue specimens for matrix-assisted laser desorption/ionization (MALDI) MSI ST:STUDY_SUMMARY that can be implemented to achieve broad coverage of central carbon, nucleotide, ST:STUDY_SUMMARY and lipid metabolism pathways. Herein, we used this approach to visualize the ST:STUDY_SUMMARY effect of nutrient stress and excess on liver metabolism. Our data revealed a ST:STUDY_SUMMARY highly organized metabolic tissue compartmentalization in livers, which becomes ST:STUDY_SUMMARY disrupted under high fat diet. Fasting caused changes in the abundance of ST:STUDY_SUMMARY several metabolites, including increased levels of fatty acids and TCA ST:STUDY_SUMMARY intermediates while fatty livers had higher levels of purine and pentose ST:STUDY_SUMMARY phosphate-related metabolites, which generate reducing equivalents to counteract ST:STUDY_SUMMARY oxidative stress. This spatially conserved approach allowed the visualization of ST:STUDY_SUMMARY liver metabolic compartmentalization at 30 µm pixel resolution and can be ST:STUDY_SUMMARY applied more broadly to yield new insights into metabolic heterogeneity in vivo. ST:INSTITUTE Brigham and Women's Hospital ST:DEPARTMENT Brigham and Women's Hospital ST:LAST_NAME Stopka ST:FIRST_NAME Sylwia ST:ADDRESS 60 Fenway Rd ST:EMAIL sstopka@bwh.harvard.edu ST:PHONE 617-525-9746 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - fed_fasted Treatment:fed_fasted RAW_FILE_NAME=fed_fasted.zip SUBJECT_SAMPLE_FACTORS - heat-freezing_treatments_liver Treatment:heat-freezing_treatments_liver RAW_FILE_NAME=heat-freezing_treatments_liver.zip SUBJECT_SAMPLE_FACTORS - High_fat_diet Treatment:High_fat_diet RAW_FILE_NAME=High_fat_diet.zip #COLLECTION CO:COLLECTION_SUMMARY C57BL/6J (000664) and BALB/cJ (000651) mice were obtained from The Jackson CO:COLLECTION_SUMMARY Laboratory. Mice were housed at 20-22°C on a 12 h light/dark cycle with ad CO:COLLECTION_SUMMARY libitum access to food (PicoLab Rodent Diet 5053) and water. All animal studies CO:COLLECTION_SUMMARY were performed in accordance with Haigis lab protocols approved by the Standing CO:COLLECTION_SUMMARY Committee on Animals, the Institutional Animal Care and Use Committee at Harvard CO:COLLECTION_SUMMARY Medical School. For heat inactivation studies, 3 mice were used (C57BL/6J, CO:COLLECTION_SUMMARY female, 7 weeks old) and kidneys, brain halves, and liver lobes from the same CO:COLLECTION_SUMMARY individual animal were subjected to the different heat inactivation treatments CO:COLLECTION_SUMMARY (overview in Supplementary Fig. 1A, E). For desiccation experiments, 2 mice were CO:COLLECTION_SUMMARY used (C57BL/6J, male, 7 weeks old). For fasting experiments, two independent CO:COLLECTION_SUMMARY cohorts of 5 mice were used per treatment group (BALB/cJ, female, 10-11 weeks CO:COLLECTION_SUMMARY old) and mice were subjected to a 16 hour overnight fast. For HFD experiments, CO:COLLECTION_SUMMARY two independent cohorts of 4 mice were used per treatment group (C57BL/6J, CO:COLLECTION_SUMMARY female). Mice were assigned at 5 weeks old to the control diet (PicoLab Rodent CO:COLLECTION_SUMMARY Diet 5053) or HFD (Research Diets, Inc. #12492) and maintained on this diet for CO:COLLECTION_SUMMARY 4.5 months. The control diet is 4.07 Gross Energy Kcal/g. The HFD is 5.21 CO:COLLECTION_SUMMARY Kcal/g. for 8-10 weeks. Comparative MALDI MSI and LC-MS analyses of tissues were CO:COLLECTION_SUMMARY always performed on the same tissue specimens. CO:SAMPLE_TYPE Liver_Brain_Kidney #TREATMENT TR:TREATMENT_SUMMARY N/A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Tissue preparation for MALDI MSI Frozen tissues were placed at -20 °C before SP:SAMPLEPREP_SUMMARY sectioning in a Microm HM550 cryostat (Thermo Scientific™). Tissues were SP:SAMPLEPREP_SUMMARY sectioned at 10 µm thickness and thaw mounted onto indium-tin-oxide SP:SAMPLEPREP_SUMMARY (ITO)-coated slides (Bruker Daltonics) for MALDI MSI analysis with serial SP:SAMPLEPREP_SUMMARY sections mounted onto glass slides for histological analyses. The microtome SP:SAMPLEPREP_SUMMARY chamber and specimen holder were maintained between -15 °C and -20 °C. Slides SP:SAMPLEPREP_SUMMARY were stored at -80 °C until further processing. For desiccation experiments, SP:SAMPLEPREP_SUMMARY slides were subjected to desiccation in a tabletop vacuum desiccator before SP:SAMPLEPREP_SUMMARY freezing. Matrix deposition A 1,5-Diaminonaphthalene(DAN)-HCl matrix solution SP:SAMPLEPREP_SUMMARY was used for all experiments. To generate the hydrochloride derivative of SP:SAMPLEPREP_SUMMARY 1,5-DAN, 39.5 mg of 1,5-DAN was dissolved in 500 µL of 1 mol/L hydrochloride SP:SAMPLEPREP_SUMMARY solution with 4 mL HPLC-grade water. The solution was sonicated for 20 minutes SP:SAMPLEPREP_SUMMARY to dissolve 1,5-DAN, after which 4.5 mL ethanol was added to yield the matrix SP:SAMPLEPREP_SUMMARY solution. Matrices were deposited on slides and tissues using a TM-sprayer (HTX SP:SAMPLEPREP_SUMMARY imaging, Carrboro, NC). DAN-HCl matrix spray conditions used where: a flow rate SP:SAMPLEPREP_SUMMARY of 0.09 mL/min, spray nozzle temperature of 75 °C, and spray nozzle velocity of SP:SAMPLEPREP_SUMMARY 1200 mm/min. A four-pass cycle was used with 2 mm track spacing and the nitrogen SP:SAMPLEPREP_SUMMARY gas pressure was maintained at 10 psi. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE None (Direct infusion) CH:INSTRUMENT_NAME timsTOF fleX CH:COLUMN_NAME none #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker timsTOF fleX MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE MALDI MS:ION_MODE NEGATIVE MS:MS_COMMENTS SCilS 2022b pro MS:MS_RESULTS_FILE ST002219_AN003628_Results.txt UNITS:Da Has m/z:Yes Has RT:No RT units:No RT data #END