Summary of Study ST003375

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002093. The data can be accessed directly via it's Project DOI: 10.21228/M82V51 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003375
Study Title	Structural and systems characterization of phosphorylation on metabolic enzymes identifies sex-specific metabolic reprogramming in obesity
Study Type	Polar metabolomics analysis of diet induced obesity
Study Summary	C57BL/6J mice were obtained from The Jackson Laboratories and housed in a specific pathogen-free facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care using laminar flow cages at 21°C. Mouse health is monitored and maintained by veterinary staff under the supervision of a veterinarian. Male and female mice fed ad libitum either a standard chow diet, a high fat diet (HFD, S3282, Bio-Serv), or a HFD supplemented with 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole (BHA, S7958, Bio-Serv) for 16 weeks beginning at age 8 weeks. All mice are euthanized at age 24 weeks following an overnight fast by inhalation of isoflurane followed by cervical dislocation, and the livers are snap frozen in liquid nitrogen upon removal.
Institute	Massachusetts Institute of Technology
Department	Biological Engineering
Laboratory	Forest White
Last Name	Tamir
First Name	Tigist
Address	500 Main st
Email	tytamir@mit.edu
Phone	5717650455
Submit Date	2024-07-30
Num Groups	4
Total Subjects	77
Num Males	39
Num Females	38
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2025-03-30
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002093
Project DOI:	doi: 10.21228/M82V51
Project Title:	Structural and systems characterization of phosphorylation on metabolic enzymes identifies sex-specific metabolic reprogramming in obesity
Project Summary:	Advances in large scale phosphoproteomics and enrichment techniques have improved our capacity to identify an increasing number of phosphorylation sites on metabolic enzymes. While some of these sites are functionally characterized, a systemwide analysis of the phosphorylation-modulated metabolic network remains largely unexplored. Moreover, there exist comprehensive genomic, epigenetic, transcriptomic, proteomic, and metabolite level annotations of metabolic networks, yet there are limited systems level analyses of how phosphorylation events on metabolic enzymes contribute to overall metabolism and network topology. In order to understand the many ways metabolic rewiring can be shaped by perturbations, we need to gain a better perspective of the combined impact of phosphorylation events on multiple metabolic enzymes and their effect on the system. Here we address this challenge by taking a systems approach that integrates computational structural analysis, phosphoproteomics, metabolomics, and biochemical validation of phosphorylation mediated regulation of metabolic enzymes. We specifically interrogate pY on metabolic enzymes using a comprehensive analysis of the in an in vivo model of high fat diet (HFD)-induced obesity where we map out metabolic tuning that alters redox homeostasis. Furthermore, we employ a targeted mass spectrometry-based approach to interrogate pathway specific phosphorylation events and apply computational modeling to identify pY sites associated with altered metabolic output. Lastly, we validate model predictions by measuring the functional role of selected pY sites using a CRISPRi-rescue system with phosphomimic or phosphodeficient enzyme variants in biochemical and isotope tracing metabolomics analysis.
Institute:	Massachusetts Institute of Technology
Department:	Biological Engineering
Laboratory:	Forest White
Last Name:	Tamir
First Name:	Tigist
Address:	500 Main st
Email:	tytamir@mit.edu
Phone:	5717650455
Funding Source:	NIH, NCI, BWF

Subject:

Subject ID:	SU003496
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL6/J
Age Or Age Range:	24 weeks
Weight Or Weight Range:	19 - 65
Gender:	Male and female
Animal Animal Supplier:	The Jackson Laboratories
Animal Housing:	housed in a specific pathogen-free facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care using laminar flow cages at 21°C. Mouse health is monitored and maintained by veterinary staff under the supervision of a veterinarian.
Animal Feed:	Male and female mice fed ad libitum either a standard chow diet, a high fat diet (HFD, S3282, Bio-Serv), or a HFD supplemented with 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole (BHA, S7958, Bio-Serv) for 16 weeks beginning at age 8 weeks.
Species Group:	Mammals

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Treatment
SA367004	ExtractionBlank	NA	Extraction Buffer + internal standards
SA367005	POOL_0-5	Pool 1-78 1 25	Pool 1-78 1 25
SA367006	POOL_0-25	Pool 1-78 1 4	Pool 1-78 1 4
SA367007	POOL_0-125	Pool 1-78 1 8	Pool 1-78 1 8
SA367008	POOL3	Pool 1-78	Pool 1-78
SA367009	POOLHFD	Pool 1-78	Pool 1-78
SA367010	POOL2	Pool 1-78	Pool 1-78
SA367011	POOLHB_0-5	Pool 79-107 1 2	Pool 79-107 1 2
SA367012	POOLHB_0-25	Pool 79-107 1 4	Pool 79-107 1 4
SA367013	POOLHB_0-125	Pool 79-107 1 8	Pool 79-107 1 8
SA367014	POOLHB	Pool 79-107	Pool 79-107
SA367015	POOLHB2	Pool 79-107	Pool 79-107
SA367016	POOLHB3	Pool 79-107	Pool 79-107
SA367017	M31	Whole Liver	High Fat Diet
SA367018	M36	Whole Liver	High Fat Diet
SA367019	M35	Whole Liver	High Fat Diet
SA367020	M34	Whole Liver	High Fat Diet
SA367021	M33	Whole Liver	High Fat Diet
SA367022	M32	Whole Liver	High Fat Diet
SA367023	M24	Whole Liver	High Fat Diet
SA367024	M30	Whole Liver	High Fat Diet
SA367025	M29	Whole Liver	High Fat Diet
SA367026	M28	Whole Liver	High Fat Diet
SA367027	M27	Whole Liver	High Fat Diet
SA367028	M26	Whole Liver	High Fat Diet
SA367029	M25	Whole Liver	High Fat Diet
SA367030	M38	Whole Liver	High Fat Diet
SA367031	M37	Whole Liver	High Fat Diet
SA367032	MB2	Whole Liver	High Fat Diet
SA367033	M39	Whole Liver	High Fat Diet
SA367034	MB4	Whole Liver	High Fat Diet
SA367035	MB10	Whole Liver	High Fat Diet
SA367036	MB9	Whole Liver	High Fat Diet
SA367037	MB8	Whole Liver	High Fat Diet
SA367038	MB7	Whole Liver	High Fat Diet
SA367039	MB6	Whole Liver	High Fat Diet
SA367040	MB5	Whole Liver	High Fat Diet
SA367041	MB3	Whole Liver	High Fat Diet
SA367042	FB1	Whole Liver	High Fat Diet
SA367043	M21	Whole Liver	High Fat Diet
SA367044	MB1	Whole Liver	High Fat Diet
SA367045	FB5	Whole Liver	High Fat Diet
SA367046	FB4	Whole Liver	High Fat Diet
SA367047	FB3	Whole Liver	High Fat Diet
SA367048	FB2	Whole Liver	High Fat Diet
SA367049	M22	Whole Liver	High Fat Diet
SA367050	M23	Whole Liver	High Fat Diet
SA367051	F36	Whole Liver	High Fat Diet
SA367052	F26	Whole Liver	High Fat Diet
SA367053	F33	Whole Liver	High Fat Diet
SA367054	F37	Whole Liver	High Fat Diet
SA367055	F38	Whole Liver	High Fat Diet
SA367056	F32	Whole Liver	High Fat Diet
SA367057	F31	Whole Liver	High Fat Diet
SA367058	F30	Whole Liver	High Fat Diet
SA367059	F29	Whole Liver	High Fat Diet
SA367060	F28	Whole Liver	High Fat Diet
SA367061	F27	Whole Liver	High Fat Diet
SA367062	F35	Whole Liver	High Fat Diet
SA367063	F34	Whole Liver	High Fat Diet
SA367064	F25	Whole Liver	High Fat Diet
SA367065	F24	Whole Liver	High Fat Diet
SA367066	F23	Whole Liver	High Fat Diet
SA367067	F22	Whole Liver	High Fat Diet
SA367068	F21	Whole Liver	High Fat Diet
SA367069	F20	Whole Liver	High Fat Diet
SA367070	FB6	Whole Liver	High Fat Diet + 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole
SA367071	MB13	Whole Liver	High Fat Diet + 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole
SA367072	FB9	Whole Liver	High Fat Diet + 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole
SA367073	FB10	Whole Liver	High Fat Diet + 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole
SA367074	MB12	Whole Liver	High Fat Diet + 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole
SA367075	FB7	Whole Liver	High Fat Diet + 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole
SA367076	FB8	Whole Liver	High Fat Diet + 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole
SA367077	MB11	Whole Liver	High Fat Diet + 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole
SA367078	MB15	Whole Liver	High Fat Diet + 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole
SA367079	MB16	Whole Liver	High Fat Diet + 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole
SA367080	MB17	Whole Liver	High Fat Diet + 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole
SA367081	MB18	Whole Liver	High Fat Diet + 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole
SA367082	MB19	Whole Liver	High Fat Diet + 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole
SA367083	MB14	Whole Liver	High Fat Diet + 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole
SA367084	F8	Whole Liver	Normal Chow Diet
SA367085	F3	Whole Liver	Normal Chow Diet
SA367086	F4	Whole Liver	Normal Chow Diet
SA367087	F5	Whole Liver	Normal Chow Diet
SA367088	F6	Whole Liver	Normal Chow Diet
SA367089	F7	Whole Liver	Normal Chow Diet
SA367090	F16	Whole Liver	Normal Chow Diet
SA367091	F9	Whole Liver	Normal Chow Diet
SA367092	F10	Whole Liver	Normal Chow Diet
SA367093	F11	Whole Liver	Normal Chow Diet
SA367094	F13	Whole Liver	Normal Chow Diet
SA367095	F14	Whole Liver	Normal Chow Diet
SA367096	F15	Whole Liver	Normal Chow Diet
SA367097	F12	Whole Liver	Normal Chow Diet
SA367098	M11	Whole Liver	Normal Chow Diet
SA367099	F17	Whole Liver	Normal Chow Diet
SA367100	M9	Whole Liver	Normal Chow Diet
SA367101	M13	Whole Liver	Normal Chow Diet
SA367102	M14	Whole Liver	Normal Chow Diet
SA367103	M15	Whole Liver	Normal Chow Diet

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Collection:

Collection ID:	CO003489
Collection Summary:	All mice are euthanized at age 24 weeks following an overnight fast by inhalation of isoflurane followed by cervical dislocation, and the livers are snap frozen in liquid nitrogen upon removal. Then livers were pulverized with Covaris cryoPREP Automated Dry Pulverizer.
Sample Type:	Liver
Collection Method:	Endpoint collection via cervical dislocation, and snap frozen liver tissue were dry pulverized
Collection Location:	UMass Chan Medical School
Collection Frequency:	NA
Collection Duration:	NA
Volumeoramount Collected:	Whole Liver
Storage Conditions:	-80℃
Collection Vials:	TUBE TT1
Storage Vials:	TUBE TT1
Collection Tube Temp:	-80
Additives:	NA

Treatment:

Treatment ID:	TR003505
Treatment Summary:	Male and female mice fed ad libitum either a standard chow diet, a high fat diet (HFD, S3282, Bio-Serv), or a HFD supplemented with 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole (BHA, S7958, Bio-Serv) for 16 weeks beginning at age 8 weeks. All mice are euthanized at age 24 weeks following an overnight fast by inhalation of isoflurane followed by cervical dislocation, and the livers are snap frozen in liquid nitrogen upon removal.

Treatment ID:

TR003505

Treatment Summary:

Male and female mice fed ad libitum either a standard chow diet, a high fat diet (HFD, S3282, Bio-Serv), or a HFD supplemented with 1.5% 2(3)‐tert‐butyl‐4 hydroxyanisole (BHA, S7958, Bio-Serv) for 16 weeks beginning at age 8 weeks. All mice are euthanized at age 24 weeks following an overnight fast by inhalation of isoflurane followed by cervical dislocation, and the livers are snap frozen in liquid nitrogen upon removal.

Sample Preparation:

Sampleprep ID:	SP003503
Sampleprep Summary:	Tissue Metabolite extraction Polar metabolites were extracted from pulverized mouse liver tissues in 800uL of 60% methanol solution and vortexed for 10min at 4oC. Then 500uL of pre-chilled chloroform was added and samples were vortexed for 10min at 4oC, followed by centrifugation at 21000xg for 10min at 4oC. The top-layer (methanol containing polar metabolites) was transferred to clean 1.7mL tubes, and the interphase (containing protein) was collected in a separate tube for protein quantitation via BCA. Polar metabolites were dried down under a nitrogen drier, and resuspended by vortexing for 10min at 4oC in HPLC-grade water containing internal standards: U-13C labeled yeast metabolite extract at 1:20 (v:v) and U-13C, 15N labeled amino acids at 500nM final concentration. Sample input was normalized during resuspension to the total protein content obtained from the BCA of the interphase layer for each sample.
Sampleprep Protocol Filename:	tytmair_5059_CollectionProtocol
Processing Storage Conditions:	-80℃
Extraction Method:	Methanol Chloroform cold extraction
Extract Storage:	-80℃
Sample Resuspension:	HPLC-grade water containing internal standards: U-13C labeled yeast metabolite extract at 1:20 (v:v) and U-13C, 15N labeled amino acids at 500nM final concentration.
Sample Spiking:	U-13C labeled yeast metabolite extract at 1:20 (v:v) and U-13C, 15N labeled amino acids at 500nM final concentration

Combined analysis:

Analysis ID	AN005527
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Vanquish
Column	SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	UNSPECIFIED
Units	area under the curve

Chromatography:

Chromatography ID:	CH004203
Chromatography Summary:	Chromatography was performed at 0.150mL/min flow rate with linear gradient of 80 – 20% of buffer B (100% Acetonitrile) for 20min, followed by 20 – 80% buffer B at 20.5min and held at 80% buffer B for the last 8min, totaling 28min in a 20mM ammonium carbonate in 0.1% ammonium hydroxide buffer A.
Instrument Name:	Thermo Vanquish
Column Name:	SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
Column Temperature:	4
Flow Gradient:	Chromatography was performed at 0.150mL/min flow rate with linear gradient of 80 – 20% of buffer B (100% Acetonitrile) for 20min, followed by 20 – 80% buffer B at 20.5min and held at 80% buffer B for the last 8min, totaling 28min in a 20mM ammonium carbonate in 0.1% ammonium hydroxide buffer A.
Flow Rate:	0.150mL/min
Solvent A:	99.9% water/0.1% ammonium hydroxide; 20mM ammonium carbonate
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS005252
Analysis ID:	AN005527
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Data was acquired on a Q Exactive Orbitrap mass spectrometer in full-scan polarity-switching mode with MS1 settings: range = 70 – 1000m/z, resolution = 70,000, AGC = 1 x 106, and maximum IT = 20ms. To maximize detection of nucleotides, an additional scan was performed in negative mode with MS1 settings: range = 200 – 700m/z, resolution = 70,000, AGC = 1 x 106, and maximum IT = 80ms. Relative quantitation of metabolites was performed with TraceFinder 2.5 a 4 mmu mass tolerance and referencing retention times from both in-house library of chemical standards as well as in-run U-13C labeled yeast metabolites.
Ion Mode:	UNSPECIFIED