Summary of Study ST001637

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 PR001047. The data can be accessed directly via it's Project DOI: 10.21228/M8C68D 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	ST001637
Study Title	A Metabolome Atlas of the Aging Mouse Brain
Study Summary	The mammalian brain relies on neurochemistry to fulfill its functions. Yet, the complexity of the brain metabolome and its changes during diseases or aging remains poorly understood. To start bridging this gap, we generated a metabolome atlas of the aging mouse brain from 10 anatomical regions spanning from adolescence to late adulthood. We combined data from three chromatography-based mass spectrometry assays and structurally annotated 1,709 metabolites to reveal the underlying architecture of aging-induced changes in the brain metabolome. Overall differences between sexes were minimal. We found 94% of all metabolites to significantly differ between brain sections in at least one age group. We also discovered that 90% of the metabolome showed significant changes with respect to age groups. For example, we identified a shift in sphingolipid patterns during aging that is related to myelin remodeling in the transition from adolescent to adult brains. This shift was accompanied by large changes in overall signature in a range of other metabolic pathways. We found clear metabolic similarities in brain sections that were functionally related such as brain stem, cerebrum and cerebellum. In cerebrum, metabolic correlation patterns got markedly weaker in the transition from adolescent to ear adults, whereas correlation patterns between cerebrum and brainstem regions decreased from early to late adulthood. We were also able to map metabolic changes to gene and protein brain atlases to link molecular changes to metabolic brain phenotypes. Metabolic profiles can be investigated via https://atlas.metabolomics.us/. This new resource enables brain researchers to link new metabolomic studies to a foundation data set.
Institute	University of California, Davis
Department	Genome Center
Laboratory	West Coast Metabolomics Center
Last Name	Ding
First Name	Jun
Address	451 East Health Science Drive, Davis, CA, 95616, USA
Email	junding@ucdavis.edu
Phone	773-326-5420
Submit Date	2020-12-23
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	GC-MS/LC-MS
Release Date	2021-08-30
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001047
Project DOI:	doi: 10.21228/M8C68D
Project Title:	A Metabolome Atlas of the Aging Mouse Brain
Project Summary:	The mammalian brain relies on neurochemistry to fulfill its functions. Yet, the complexity of the brain metabolome and its changes during diseases or aging remains poorly understood. To start bridging this gap, we generated a metabolome atlas of the aging mouse brain from 10 anatomical regions spanning from adolescence to late adulthood. We combined data from three chromatography-based mass spectrometry assays and structurally annotated 1,709 metabolites to reveal the underlying architecture of aging-induced changes in the brain metabolome. Overall differences between sexes were minimal. We found 94% of all metabolites to significantly differ between brain sections in at least one age group. We also discovered that 90% of the metabolome showed significant changes with respect to age groups. For example, we identified a shift in sphingolipid patterns during aging that is related to myelin remodeling in the transition from adolescent to adult brains. This shift was accompanied by large changes in overall signature in a range of other metabolic pathways. We found clear metabolic similarities in brain sections that were functionally related such as brain stem, cerebrum and cerebellum. In cerebrum, metabolic correlation patterns got markedly weaker in the transition from adolescent to ear adults, whereas correlation patterns between cerebrum and brainstem regions decreased from early to late adulthood. We were also able to map metabolic changes to gene and protein brain atlases to link molecular changes to metabolic brain phenotypes. Metabolic profiles can be investigated via https://atlas.metabolomics.us/. This new resource enables brain researchers to link new metabolomic studies to a foundation data set.
Institute:	University of California, Davis
Department:	Genome Center
Laboratory:	West Coast Metabolomics Center
Last Name:	Ding
First Name:	Jun
Address:	451 East Health Science Drive, Davis, CA, 95616, USA
Email:	junding@ucdavis.edu
Phone:	773-326-5420
Funding Source:	NIH U2C ES030158

Subject:

Subject ID:	SU001714
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6NCrl
Age Or Age Range:	3 weeks, 16 weeks and 59 weeks old
Gender:	Male and female

Factors:

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

mb_sample_id	local_sample_id	Brain region	Age	Gender
SA149974	EA_F4_BG	Basal ganglia	16 weeks	Female
SA149975	EA_F3_BG	Basal ganglia	16 weeks	Female
SA149976	EA_F5_BG	Basal ganglia	16 weeks	Female
SA149977	EA_F7_BG	Basal ganglia	16 weeks	Female
SA149978	EA_F8_BG	Basal ganglia	16 weeks	Female
SA149979	EA_F1_BG	Basal ganglia	16 weeks	Female
SA149980	EA_F6_BG	Basal ganglia	16 weeks	Female
SA149981	EA_F2_BG	Basal ganglia	16 weeks	Female
SA149982	EA_M4_BG	Basal ganglia	16 weeks	Male
SA149983	EA_M3_BG	Basal ganglia	16 weeks	Male
SA149984	EA_M2_BG	Basal ganglia	16 weeks	Male
SA149985	EA_M1_BG	Basal ganglia	16 weeks	Male
SA149986	EA_M5_BG	Basal ganglia	16 weeks	Male
SA149987	EA_M6_BG	Basal ganglia	16 weeks	Male
SA149988	EA_M7_BG	Basal ganglia	16 weeks	Male
SA149989	EA_M8_BG	Basal ganglia	16 weeks	Male
SA149990	AD_F3_BG	Basal ganglia	3 weeks	Female
SA149991	AD_F2_BG	Basal ganglia	3 weeks	Female
SA149992	AD_F1_BG	Basal ganglia	3 weeks	Female
SA149993	AD_F5_BG	Basal ganglia	3 weeks	Female
SA149994	AD_F4_BG	Basal ganglia	3 weeks	Female
SA149995	AD_F7_BG	Basal ganglia	3 weeks	Female
SA149996	AD_F8_BG	Basal ganglia	3 weeks	Female
SA149997	AD_F6_BG	Basal ganglia	3 weeks	Female
SA149998	AD_M5_BG	Basal ganglia	3 weeks	Male
SA149999	AD_M4_BG	Basal ganglia	3 weeks	Male
SA150000	AD_M3_BG	Basal ganglia	3 weeks	Male
SA150001	AD_M6_BG	Basal ganglia	3 weeks	Male
SA150002	AD_M8_BG	Basal ganglia	3 weeks	Male
SA150003	AD_M7_BG	Basal ganglia	3 weeks	Male
SA150004	AD_M1_BG	Basal ganglia	3 weeks	Male
SA150005	AD_M2_BG	Basal ganglia	3 weeks	Male
SA150006	LA_F7_BG	Basal ganglia	59 weeks	Female
SA150007	LA_F6_BG	Basal ganglia	59 weeks	Female
SA150008	LA_F8_BG	Basal ganglia	59 weeks	Female
SA150009	LA_F1_BG	Basal ganglia	59 weeks	Female
SA150010	LA_F2_BG	Basal ganglia	59 weeks	Female
SA150011	LA_F3_BG	Basal ganglia	59 weeks	Female
SA150012	LA_F5_BG	Basal ganglia	59 weeks	Female
SA150013	LA_F4_BG	Basal ganglia	59 weeks	Female
SA150014	LA_M6_BG	Basal ganglia	59 weeks	Male
SA150015	LA_M8_BG	Basal ganglia	59 weeks	Male
SA150016	LA_M5_BG	Basal ganglia	59 weeks	Male
SA150017	LA_M7_BG	Basal ganglia	59 weeks	Male
SA150018	LA_M4_BG	Basal ganglia	59 weeks	Male
SA150019	LA_M2_BG	Basal ganglia	59 weeks	Male
SA150020	LA_M1_BG	Basal ganglia	59 weeks	Male
SA150021	LA_M3_BG	Basal ganglia	59 weeks	Male
SA150022	EA_F5_CB	Cerebellum	16 weeks	Female
SA150023	EA_F6_CB	Cerebellum	16 weeks	Female
SA150024	EA_F7_CB	Cerebellum	16 weeks	Female
SA150025	EA_F4_CB	Cerebellum	16 weeks	Female
SA150026	EA_F3_CB	Cerebellum	16 weeks	Female
SA150027	EA_F2_CB	Cerebellum	16 weeks	Female
SA150028	EA_F8_CB	Cerebellum	16 weeks	Female
SA150029	EA_F1_CB	Cerebellum	16 weeks	Female
SA150030	EA_M8_CB	Cerebellum	16 weeks	Male
SA150031	EA_M6_CB	Cerebellum	16 weeks	Male
SA150032	EA_M7_CB	Cerebellum	16 weeks	Male
SA150033	EA_M5_CB	Cerebellum	16 weeks	Male
SA150034	EA_M4_CB	Cerebellum	16 weeks	Male
SA150035	EA_M2_CB	Cerebellum	16 weeks	Male
SA150036	EA_M3_CB	Cerebellum	16 weeks	Male
SA150037	EA_M1_CB	Cerebellum	16 weeks	Male
SA150038	AD_F8_CB	Cerebellum	3 weeks	Female
SA150039	AD_F6_CB	Cerebellum	3 weeks	Female
SA150040	AD_F7_CB	Cerebellum	3 weeks	Female
SA150041	AD_F5_CB	Cerebellum	3 weeks	Female
SA150042	AD_F3_CB	Cerebellum	3 weeks	Female
SA150043	AD_F2_CB	Cerebellum	3 weeks	Female
SA150044	AD_F1_CB	Cerebellum	3 weeks	Female
SA150045	AD_F4_CB	Cerebellum	3 weeks	Female
SA150046	AD_M6_CB	Cerebellum	3 weeks	Male
SA150047	AD_M8_CB	Cerebellum	3 weeks	Male
SA150048	AD_M1_CB	Cerebellum	3 weeks	Male
SA150049	AD_M5_CB	Cerebellum	3 weeks	Male
SA150050	AD_M7_CB	Cerebellum	3 weeks	Male
SA150051	AD_M2_CB	Cerebellum	3 weeks	Male
SA150052	AD_M3_CB	Cerebellum	3 weeks	Male
SA150053	AD_M4_CB	Cerebellum	3 weeks	Male
SA150054	LA_F7_CB	Cerebellum	59 weeks	Female
SA150055	LA_F8_CB	Cerebellum	59 weeks	Female
SA150056	LA_F6_CB	Cerebellum	59 weeks	Female
SA150057	LA_F3_CB	Cerebellum	59 weeks	Female
SA150058	LA_F5_CB	Cerebellum	59 weeks	Female
SA150059	LA_F2_CB	Cerebellum	59 weeks	Female
SA150060	LA_F1_CB	Cerebellum	59 weeks	Female
SA150061	LA_F4_CB	Cerebellum	59 weeks	Female
SA150062	LA_M7_CB	Cerebellum	59 weeks	Male
SA150063	LA_M6_CB	Cerebellum	59 weeks	Male
SA150064	LA_M8_CB	Cerebellum	59 weeks	Male
SA150065	LA_M1_CB	Cerebellum	59 weeks	Male
SA150066	LA_M5_CB	Cerebellum	59 weeks	Male
SA150067	LA_M3_CB	Cerebellum	59 weeks	Male
SA150068	LA_M2_CB	Cerebellum	59 weeks	Male
SA150069	LA_M4_CB	Cerebellum	59 weeks	Male
SA150070	EA_F6_CT	Cerebral cortex	16 weeks	Female
SA150071	EA_F7_CT	Cerebral cortex	16 weeks	Female
SA150072	EA_F5_CT	Cerebral cortex	16 weeks	Female
SA150073	EA_F2_CT	Cerebral cortex	16 weeks	Female

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Collection:

Collection ID:	CO001707
Collection Summary:	Brain tissue samples were collected from 3 weeks, 16 weeks and 59 weeks old male and female wild type mice on a C57BL/6NCrl background and performed under approved institutional IACUC protocols. Briefly, mice were anesthetized with 4% Isoflurane in 100% oxygen at a flow rate of 3 L/h to a surgical plane. Blood was then collected by retro-orbital bleed into an EDTA tube and centrifuged at 3000 rpm for 15 min to separate and remove plasma. While under anesthsia mice were perfused for approximately 10 minutes with phosphate buffered saline (PBS) pH 7.4 at room temperature. Following perfusion, the brain was removed and placed in a petri dish containing PBS at 4oC for dissection of individual brain regions. A dissection microscope, fine tip (#5) forceps, and razor blade was used to isolate and separate brain regions (olfactory bulb, hippocampus, hypothalamus, thalamus, midbrain, cerebellum, pons, medulla, cerebral cortex, and basal ganglia collected as caudate putamen and basal forebrain) in induvial mice while being careful to avoid contamination from neighboring regions. Briefly, after separating the olfactory bulbs, the left and right cerebral cortices were then removed while taking care not to disrupt the regions underneath. This enabled access to and removal of the left and right hippocampus. After cutting along the thalamus, the left and right caudate putamen was separated and removed from the basal forebrain. Subsequently, the cerebellum and midbrain were isolated and removed, followed by separation and removal of the thalamus and the hypothalamus from the pons and medulla. The pons was then separated from the medulla. Any spinal cord remaining on the medulla was removed. Each region was immediately placed in a cryo vial and flash frozen in liquid nitrogen for analysis.
Sample Type:	Brain

Treatment:

Treatment ID:	TR001727
Treatment Summary:	Five milligrams of tissue from each brain region were homogenized in 225 µL of -20oC cold, internal standard-containing methanol using a GenoGrinder 2010 (SPEX SamplePrep) for 2 min at 1,350 rpm. The homogenate was vortexed for 10 s. 750 µL of -20oC cold, internal standard-containing methyl tertiary-butyl ether (MTBE) was added, and the mixture was vortexed for 10 s and shaken at 4 oC for 5 min with an Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments). MTBE contained cholesteryl ester 22:1 as internal standard. Next, 188 µL room temperature water was added and vortexed for 20 s to induce phase separation. After centrifugation for 2 min at 14,000 g, two 350 µL aliquots of the upper non-polar phase and two 125 µL aliquots of the bottom polar phase were collected and dried down. Remaining fractions were combined to form QC pools and were injected after every set of 10 biological samples. The non-polar phase employed for lipidomics was resuspended in a mixture of methanol/toluene (60 µL, 9:1, v/v) containing an internal standard [12-[(cyclohexylamino) carbonyl]amino]-dodecanoic acid (CUDA)] before injection. Resuspension of dried polar phases for HILIC analysis was performed in a mixture of internal standard-containing acetonitrile/water (90 µL, 4:1, v/v). The second dried polar phase was reserved for GC analysis and a following derivatization process was carried out before injection. First, carbonyl groups were protected by methoximation with methoxyamine hydrochloride in pyridine (40 mg/mL, 10 µL) was added to the dried samples. Then, the mixture was incubated at 30˚C for 90 min followed by trimethylsilylation with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA, 90 μL) containing C8–C30 fatty acid methyl esters (FAMEs) as internal standards by shaking at 37˚C for 30 min.

Treatment ID:

TR001727

Treatment Summary:

Five milligrams of tissue from each brain region were homogenized in 225 µL of -20oC cold, internal standard-containing methanol using a GenoGrinder 2010 (SPEX SamplePrep) for 2 min at 1,350 rpm. The homogenate was vortexed for 10 s. 750 µL of -20oC cold, internal standard-containing methyl tertiary-butyl ether (MTBE) was added, and the mixture was vortexed for 10 s and shaken at 4 oC for 5 min with an Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments). MTBE contained cholesteryl ester 22:1 as internal standard. Next, 188 µL room temperature water was added and vortexed for 20 s to induce phase separation. After centrifugation for 2 min at 14,000 g, two 350 µL aliquots of the upper non-polar phase and two 125 µL aliquots of the bottom polar phase were collected and dried down. Remaining fractions were combined to form QC pools and were injected after every set of 10 biological samples. The non-polar phase employed for lipidomics was resuspended in a mixture of methanol/toluene (60 µL, 9:1, v/v) containing an internal standard [12-[(cyclohexylamino) carbonyl]amino]-dodecanoic acid (CUDA)] before injection. Resuspension of dried polar phases for HILIC analysis was performed in a mixture of internal standard-containing acetonitrile/water (90 µL, 4:1, v/v). The second dried polar phase was reserved for GC analysis and a following derivatization process was carried out before injection. First, carbonyl groups were protected by methoximation with methoxyamine hydrochloride in pyridine (40 mg/mL, 10 µL) was added to the dried samples. Then, the mixture was incubated at 30˚C for 90 min followed by trimethylsilylation with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA, 90 μL) containing C8–C30 fatty acid methyl esters (FAMEs) as internal standards by shaking at 37˚C for 30 min.

Sample Preparation:

Sampleprep ID:	SP001720
Sampleprep Summary:	Five milligrams of tissue from each brain region were homogenized in 225 µL of -20oC cold, internal standard-containing methanol using a GenoGrinder 2010 (SPEX SamplePrep) for 2 min at 1,350 rpm. The homogenate was vortexed for 10 s. 750 µL of -20oC cold, internal standard-containing methyl tertiary-butyl ether (MTBE) was added, and the mixture was vortexed for 10 s and shaken at 4 oC for 5 min with an Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments). MTBE contained cholesteryl ester 22:1 as internal standard. Next, 188 µL room temperature water was added and vortexed for 20 s to induce phase separation. After centrifugation for 2 min at 14,000 g, two 350 µL aliquots of the upper non-polar phase and two 125 µL aliquots of the bottom polar phase were collected and dried down. Remaining fractions were combined to form QC pools and were injected after every set of 10 biological samples. The non-polar phase employed for lipidomics was resuspended in a mixture of methanol/toluene (60 µL, 9:1, v/v) containing an internal standard [12-[(cyclohexylamino) carbonyl]amino]-dodecanoic acid (CUDA)] before injection. Resuspension of dried polar phases for HILIC analysis was performed in a mixture of internal standard-containing acetonitrile/water (90 µL, 4:1, v/v). The second dried polar phase was reserved for GC analysis and a following derivatization process was carried out before injection. First, carbonyl groups were protected by methoximation with methoxyamine hydrochloride in pyridine (40 mg/mL, 10 µL) was added to the dried samples. Then, the mixture was incubated at 30˚C for 90 min followed by trimethylsilylation with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA, 90 μL) containing C8–C30 fatty acid methyl esters (FAMEs) as internal standards by shaking at 37˚C for 30 min.

Sampleprep ID:

SP001720

Sampleprep Summary:

Combined analysis:

Analysis ID	AN002675	AN002676	AN002677	AN002678	AN002679
Analysis type	MS	MS	MS	MS	MS
Chromatography type	HILIC	HILIC	Reversed phase	Reversed phase	GC
Chromatography system	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish	Agilent 6890N
Column	Waters XBridge Amide (100 x 4.6mm,3.5um)	Waters XBridge Amide (100 x 4.6mm,3.5um)	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)	Restek Rtx-5Sil (30m x 0.25mm,0.25um)
MS Type	ESI	ESI	ESI	ESI	EI
MS instrument type	Orbitrap	LTQ-FT	Orbitrap	Ion trap	GC-TOF
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Leco Pegasus IV TOF
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE	POSITIVE
Units	Peak height	Peak height	Peak height	Peak height	Peak height

Chromatography:

Chromatography ID:	CH001969
Chromatography Summary:	HILIC positive
Instrument Name:	Thermo Vanquish
Column Name:	Waters XBridge Amide (100 x 4.6mm,3.5um)
Chromatography Type:	HILIC

Chromatography ID:	CH001970
Chromatography Summary:	HILIC negative
Instrument Name:	Thermo Vanquish
Column Name:	Waters XBridge Amide (100 x 4.6mm,3.5um)
Chromatography Type:	HILIC

Chromatography ID:	CH001971
Chromatography Summary:	CSH positive
Instrument Name:	Thermo Vanquish
Column Name:	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
Chromatography Type:	Reversed phase

Chromatography ID:	CH001972
Chromatography Summary:	CSH negative
Instrument Name:	Thermo Vanquish
Column Name:	Waters Acquity CSH C18 (100 x 2.1mm,1.7um)
Chromatography Type:	Reversed phase

Chromatography ID:	CH001973
Chromatography Summary:	GC
Instrument Name:	Agilent 6890N
Column Name:	Restek Rtx-5Sil (30m x 0.25mm,0.25um)
Chromatography Type:	GC

MS:

MS ID:	MS002474
Analysis ID:	AN002675
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The ion source conditions were set as follows: spray voltage, 3.6 kV; sheath gas flow rate, 60 arbitrary units; aux gas flow rate, 25 arbitrary units; sweep gas flow rate, 2 arbitrary units; capillary temp, 300 °C; S-lens RF level, 50; Aux gas heater temp, 370 °C. The following acquisition parameters were used for MS1 analysis: resolution, 60000, AGC target, 1e6; Maximum IT, 100 ms; scan range 60-900 m/z; spectrum data type, centroid. Data dependent MS/MS parameters: resolution, 15000; AGC target, 1e5; maximum IT, 50 ms; loop count, 4; TopN, 4; isolation window, 1.0 m/z; fixed first mass, 70.0 m/z; (N)CE/ stepped nce, 20, 30, 40; spectrum data type, centroid; minimum AGC target, 8e3; intensity threshold, 1.6e5; exclude isotopes, on; dynamic exclusion, 3.0 s. To increase the total number of MS/MS spectra, five runs with iterative MS/MS exclusions were performed using the R package “IE-Omics”18 for both positive and negative electrospray conditions. All the LC-MS raw data files were converted into ABF format using ABF converter (https://www.reifycs.com/AbfConverter/). MS-DIAL ver.4.00 software was used for deconvolution, peak picking, alignment, and compound identification19. The detailed parameter setting was as follows: MS1 tolerance, 0.005 Da; MS2 tolerance, 0.01 Da; minimum peak height, 20000 amplitude; mass slice width, 0.1 Da; smoothing method, linear weighted moving average; smoothing level, 5 scans; minimum peak width, 10 scans. [M+H]+, [M+NH4]+, [M+Na]+, [2M+H]+,[2M+NH4]+, [2M+Na]+ were included in adduct ion setting for positive mode lipidomics and HILIC analysis, [M-H]-, [M+Cl]-, [M+Hac-H]- for negative mode lipidomics, and [M-H]-, [M+Cl]-, [M+FA-H]-, [2M-H]- for negative mode HILIC analysis. Compounds were annotated by matching retention times, accurate precursor masses and MS/MS spectra against libraries in MassBank of North America and NIST17. Retention time libraries were produced from authentic standards and extrapolated for lipids as published before. The primary result data matrix was processed with MS-FLO software to identify ion adducts, duplicate peaks, and isotopic features. Systematic error removal by random forest (SERRF software) was employed to correct for batch effects or instrument signal drifts. Statistical analysis was performed by normalization to the median intensity of all identified compounds, log transformation and Pareto scaling. PCA was used for multivariate statistics and visualization, specifically for outlier detection. Two outliers, including one medulla sample from a female early adult and one basal ganglia sample from a female late adult, were removed. Results from Kruskal-Wallis tests were followed by Dunn’s multiple comparison confinement. Results from Mann–Whitney U tests were corrected by the Benjamini–Hochberg procedure to control the false discovery rate. Spearman rank correlation analyses and fold change calculations were conducted using R.
Ion Mode:	POSITIVE

MS ID:	MS002475
Analysis ID:	AN002676
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	LTQ-FT
MS Type:	ESI
MS Comments:	The ion source conditions were set as follows: spray voltage, -3.0 kV; sheath gas flow rate, 60 arbitrary units; aux gas flow rate, 25 arbitrary units; sweep gas flow rate, 2 arbitrary units; capillary temp, 300 °C; S-lens RF level, 50; Aux gas heater temp, 370 °C. The following acquisition parameters were used for MS1 analysis: resolution, 60000, AGC target, 1e6; Maximum IT, 100 ms; scan range 60-900 m/z; spectrum data type, centroid. Data dependent MS/MS parameters: resolution, 15000; AGC target, 1e5; maximum IT, 50 ms; loop count, 4; TopN, 4; isolation window, 1.0 m/z; fixed first mass, 70.0 m/z; (N)CE/ stepped nce, 20, 30, 40; spectrum data type, centroid; minimum AGC target, 8e3; intensity threshold, 1.6e5; exclude isotopes, on; dynamic exclusion, 3.0 s. To increase the total number of MS/MS spectra, five runs with iterative MS/MS exclusions were performed using the R package “IE-Omics”18 for both positive and negative electrospray conditions. All the LC-MS raw data files were converted into ABF format using ABF converter (https://www.reifycs.com/AbfConverter/). MS-DIAL ver.4.00 software was used for deconvolution, peak picking, alignment, and compound identification19. The detailed parameter setting was as follows: MS1 tolerance, 0.005 Da; MS2 tolerance, 0.01 Da; minimum peak height, 20000 amplitude; mass slice width, 0.1 Da; smoothing method, linear weighted moving average; smoothing level, 5 scans; minimum peak width, 10 scans. [M+H]+, [M+NH4]+, [M+Na]+, [2M+H]+,[2M+NH4]+, [2M+Na]+ were included in adduct ion setting for positive mode lipidomics and HILIC analysis, [M-H]-, [M+Cl]-, [M+Hac-H]- for negative mode lipidomics, and [M-H]-, [M+Cl]-, [M+FA-H]-, [2M-H]- for negative mode HILIC analysis. Compounds were annotated by matching retention times, accurate precursor masses and MS/MS spectra against libraries in MassBank of North America and NIST17. Retention time libraries were produced from authentic standards and extrapolated for lipids as published before. The primary result data matrix was processed with MS-FLO software to identify ion adducts, duplicate peaks, and isotopic features. Systematic error removal by random forest (SERRF software) was employed to correct for batch effects or instrument signal drifts. Statistical analysis was performed by normalization to the median intensity of all identified compounds, log transformation and Pareto scaling. PCA was used for multivariate statistics and visualization, specifically for outlier detection. Two outliers, including one medulla sample from a female early adult and one basal ganglia sample from a female late adult, were removed. Results from Kruskal-Wallis tests were followed by Dunn’s multiple comparison confinement. Results from Mann–Whitney U tests were corrected by the Benjamini–Hochberg procedure to control the false discovery rate. Spearman rank correlation analyses and fold change calculations were conducted using R.
Ion Mode:	NEGATIVE

MS ID:	MS002476
Analysis ID:	AN002677
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The ion source conditions were set as follows: spray voltage, 3.6 kV; sheath gas flow rate, 60 arbitrary units; aux gas flow rate, 25 arbitrary units; sweep gas flow rate, 2 arbitrary units; capillary temp, 300 °C; S-lens RF level, 50; Aux gas heater temp, 370 °C. The following acquisition parameters were used for MS1 analysis: resolution, 60000, AGC target, 1e6; Maximum IT, 100 ms; scan range 150-1700 m/z; spectrum data type, centroid. Data dependent MS/MS parameters: resolution, 15000; AGC target, 1e5; maximum IT, 50 ms; loop count, 4; TopN, 4; isolation window, 1.0 m/z; fixed first mass, 70.0 m/z; (N)CE/ stepped nce, 20, 30, 40; spectrum data type, centroid; minimum AGC target, 8e3; intensity threshold, 1.6e5; exclude isotopes, on; dynamic exclusion, 3.0 s. To increase the total number of MS/MS spectra, five runs with iterative MS/MS exclusions were performed using the R package “IE-Omics”18 for both positive and negative electrospray conditions. All the LC-MS raw data files were converted into ABF format using ABF converter (https://www.reifycs.com/AbfConverter/). MS-DIAL ver.4.00 software was used for deconvolution, peak picking, alignment, and compound identification19. The detailed parameter setting was as follows: MS1 tolerance, 0.005 Da; MS2 tolerance, 0.01 Da; minimum peak height, 20000 amplitude; mass slice width, 0.1 Da; smoothing method, linear weighted moving average; smoothing level, 5 scans; minimum peak width, 10 scans. [M+H]+, [M+NH4]+, [M+Na]+, [2M+H]+,[2M+NH4]+, [2M+Na]+ were included in adduct ion setting for positive mode lipidomics and HILIC analysis, [M-H]-, [M+Cl]-, [M+Hac-H]- for negative mode lipidomics, and [M-H]-, [M+Cl]-, [M+FA-H]-, [2M-H]- for negative mode HILIC analysis. Compounds were annotated by matching retention times, accurate precursor masses and MS/MS spectra against libraries in MassBank of North America and NIST17. Retention time libraries were produced from authentic standards and extrapolated for lipids as published before. The primary result data matrix was processed with MS-FLO software to identify ion adducts, duplicate peaks, and isotopic features. Systematic error removal by random forest (SERRF software) was employed to correct for batch effects or instrument signal drifts. Statistical analysis was performed by normalization to the median intensity of all identified compounds, log transformation and Pareto scaling. PCA was used for multivariate statistics and visualization, specifically for outlier detection. Two outliers, including one medulla sample from a female early adult and one basal ganglia sample from a female late adult, were removed. Results from Kruskal-Wallis tests were followed by Dunn’s multiple comparison confinement. Results from Mann–Whitney U tests were corrected by the Benjamini–Hochberg procedure to control the false discovery rate. Spearman rank correlation analyses and fold change calculations were conducted using R.
Ion Mode:	POSITIVE

MS ID:	MS002477
Analysis ID:	AN002678
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Ion trap
MS Type:	ESI
MS Comments:	The ion source conditions were set as follows: spray voltage, -3.0 kV; sheath gas flow rate, 60 arbitrary units; aux gas flow rate, 25 arbitrary units; sweep gas flow rate, 2 arbitrary units; capillary temp, 300 °C; S-lens RF level, 50; Aux gas heater temp, 370 °C. The following acquisition parameters were used for MS1 analysis: resolution, 60000, AGC target, 1e6; Maximum IT, 100 ms; scan range 150-1700 m/z; spectrum data type, centroid. Data dependent MS/MS parameters: resolution, 15000; AGC target, 1e5; maximum IT, 50 ms; loop count, 4; TopN, 4; isolation window, 1.0 m/z; fixed first mass, 70.0 m/z; (N)CE/ stepped nce, 20, 30, 40; spectrum data type, centroid; minimum AGC target, 8e3; intensity threshold, 1.6e5; exclude isotopes, on; dynamic exclusion, 3.0 s. To increase the total number of MS/MS spectra, five runs with iterative MS/MS exclusions were performed using the R package “IE-Omics”18 for both positive and negative electrospray conditions. All the LC-MS raw data files were converted into ABF format using ABF converter (https://www.reifycs.com/AbfConverter/). MS-DIAL ver.4.00 software was used for deconvolution, peak picking, alignment, and compound identification19. The detailed parameter setting was as follows: MS1 tolerance, 0.005 Da; MS2 tolerance, 0.01 Da; minimum peak height, 20000 amplitude; mass slice width, 0.1 Da; smoothing method, linear weighted moving average; smoothing level, 5 scans; minimum peak width, 10 scans. [M+H]+, [M+NH4]+, [M+Na]+, [2M+H]+,[2M+NH4]+, [2M+Na]+ were included in adduct ion setting for positive mode lipidomics and HILIC analysis, [M-H]-, [M+Cl]-, [M+Hac-H]- for negative mode lipidomics, and [M-H]-, [M+Cl]-, [M+FA-H]-, [2M-H]- for negative mode HILIC analysis. Compounds were annotated by matching retention times, accurate precursor masses and MS/MS spectra against libraries in MassBank of North America and NIST17. Retention time libraries were produced from authentic standards and extrapolated for lipids as published before. The primary result data matrix was processed with MS-FLO software to identify ion adducts, duplicate peaks, and isotopic features. Systematic error removal by random forest (SERRF software) was employed to correct for batch effects or instrument signal drifts. Statistical analysis was performed by normalization to the median intensity of all identified compounds, log transformation and Pareto scaling. PCA was used for multivariate statistics and visualization, specifically for outlier detection. Two outliers, including one medulla sample from a female early adult and one basal ganglia sample from a female late adult, were removed. Results from Kruskal-Wallis tests were followed by Dunn’s multiple comparison confinement. Results from Mann–Whitney U tests were corrected by the Benjamini–Hochberg procedure to control the false discovery rate. Spearman rank correlation analyses and fold change calculations were conducted using R.
Ion Mode:	NEGATIVE

MS ID:	MS002478
Analysis ID:	AN002679
Instrument Name:	Leco Pegasus IV TOF
Instrument Type:	GC-TOF
MS Type:	EI
MS Comments:	0.5 μL sample was injected with 25 s splitless time on an Agilent 6890 GC (Agilent Technologies, Santa Clara, CA) using a Restek Rtx-5Sil MS column (30 m x 0.25 mm, 0.25 μm) with 10 m Guard column (10 m x 0.25 mm, 0.25 μm) and 1 mL/min Helium gas flow. Oven temperature was held 50°C for 1 min, ramped up to 330 °C at 20 °C/min and held for 5 min. Data was acquired at 70 eV electron ionization at 17 spectra/s from 85 to 500 Da at 1850 V detector voltage on a Leco Pegasus IV time-of-flight mass spectrometer (Leco Corporation, St. Joseph, MI). The transfer line temperature was held at 280 °C with an ion source temperature set at 250 °C. Standard metabolites mixtures and blank samples were injected at the beginning of the run and every ten samples throughout the run for quality control. Raw data was preprocessed by ChromaTOF version 4.50 for baseline subtraction, deconvolution and peak detection. Binbase was used for metabolite annotation and reporting.
Ion Mode:	POSITIVE