#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