Summary of Study ST001888

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	ST001888
Study Title	A Metabolome Atlas of the Aging Mouse Brain (Study part II)
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 wildtype male and female mouse brain from 10 anatomical regions spanning from adolescence to old age. We combined data from three chromatography-based mass spectrometry assays and structurally annotated 1,547 metabolites to reveal the underlying architecture of aging-induced changes in the brain metabolome. Overall differences between sexes were minimal. We found 99% of all metabolites to significantly differ between brain regions in at least one age group. We also discovered that 97% 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 aging 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 regions 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 adulthood, whereas the overall correlation patterns between all regions reflected a decreased brain segregation at old age. 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://mouse.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	2021-07-25
Raw Data Available	Yes
Raw Data File Type(s)	cdf, 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:	SU001966
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6NCrl
Age Or Age Range:	92 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	Gender
SA175310	900483-015-165	Basal ganglia	Female
SA175311	900483-010-110	Basal ganglia	Female
SA175312	900483-011-121	Basal ganglia	Female
SA175313	900483-016-176	Basal ganglia	Female
SA175314	900483-014-154	Basal ganglia	Female
SA175315	900483-009-099	Basal ganglia	Female
SA175316	900483-013-143	Basal ganglia	Female
SA175317	900483-012-132	Basal ganglia	Female
SA175318	900483-014-066	Basal ganglia	Male
SA175319	900483-011-033	Basal ganglia	Male
SA175320	900483-015-077	Basal ganglia	Male
SA175321	900483-012-044	Basal ganglia	Male
SA175322	900483-016-088	Basal ganglia	Male
SA175323	900483-013-055	Basal ganglia	Male
SA175324	900483-010-022	Basal ganglia	Male
SA175325	900483-009-011	Basal ganglia	Male
SA175326	900483-010-103	Cerebellum	Female
SA175327	900483-011-114	Cerebellum	Female
SA175328	900483-013-136	Cerebellum	Female
SA175329	900483-012-125	Cerebellum	Female
SA175330	900483-014-147	Cerebellum	Female
SA175331	900483-016-169	Cerebellum	Female
SA175332	900483-015-158	Cerebellum	Female
SA175333	900483-009-092	Cerebellum	Female
SA175334	900483-013-048	Cerebellum	Male
SA175335	900483-011-026	Cerebellum	Male
SA175336	900483-009-004	Cerebellum	Male
SA175337	900483-010-015	Cerebellum	Male
SA175338	900483-015-070	Cerebellum	Male
SA175339	900483-016-081	Cerebellum	Male
SA175340	900483-012-037	Cerebellum	Male
SA175341	900483-014-059	Cerebellum	Male
SA175342	900483-009-090	Cerebral cortex	Female
SA175343	900483-012-123	Cerebral cortex	Female
SA175344	900483-011-112	Cerebral cortex	Female
SA175345	900483-014-145	Cerebral cortex	Female
SA175346	900483-016-167	Cerebral cortex	Female
SA175347	900483-015-156	Cerebral cortex	Female
SA175348	900483-013-134	Cerebral cortex	Female
SA175349	900483-010-101	Cerebral cortex	Female
SA175350	900483-016-079	Cerebral cortex	Male
SA175351	900483-010-013	Cerebral cortex	Male
SA175352	900483-013-046	Cerebral cortex	Male
SA175353	900483-012-035	Cerebral cortex	Male
SA175354	900483-014-057	Cerebral cortex	Male
SA175355	900483-009-002	Cerebral cortex	Male
SA175356	900483-011-024	Cerebral cortex	Male
SA175357	900483-015-068	Cerebral cortex	Male
SA175358	900483-013-135	Hippocampus	Female
SA175359	900483-010-102	Hippocampus	Female
SA175360	900483-012-124	Hippocampus	Female
SA175361	900483-009-091	Hippocampus	Female
SA175362	900483-014-146	Hippocampus	Female
SA175363	900483-015-157	Hippocampus	Female
SA175364	900483-011-113	Hippocampus	Female
SA175365	900483-016-168	Hippocampus	Female
SA175366	900483-009-003	Hippocampus	Male
SA175367	900483-013-047	Hippocampus	Male
SA175368	900483-011-025	Hippocampus	Male
SA175369	900483-014-058	Hippocampus	Male
SA175370	900483-012-036	Hippocampus	Male
SA175371	900483-016-080	Hippocampus	Male
SA175372	900483-015-069	Hippocampus	Male
SA175373	900483-010-014	Hippocampus	Male
SA175374	900483-012-128	Hypothalamus	Female
SA175375	900483-015-161	Hypothalamus	Female
SA175376	900483-013-139	Hypothalamus	Female
SA175377	900483-011-117	Hypothalamus	Female
SA175378	900483-010-106	Hypothalamus	Female
SA175379	900483-016-172	Hypothalamus	Female
SA175380	900483-009-095	Hypothalamus	Female
SA175381	900483-014-150	Hypothalamus	Female
SA175382	900483-013-051	Hypothalamus	Male
SA175383	900483-014-062	Hypothalamus	Male
SA175384	900483-009-007	Hypothalamus	Male
SA175385	900483-012-040	Hypothalamus	Male
SA175386	900483-011-029	Hypothalamus	Male
SA175387	900483-015-073	Hypothalamus	Male
SA175388	900483-010-018	Hypothalamus	Male
SA175389	900483-016-084	Hypothalamus	Male
SA175390	900483-013-141	Medulla	Female
SA175391	900483-010-108	Medulla	Female
SA175392	900483-016-174	Medulla	Female
SA175393	900483-011-119	Medulla	Female
SA175394	900483-009-097	Medulla	Female
SA175395	900483-012-130	Medulla	Female
SA175396	900483-014-152	Medulla	Female
SA175397	900483-015-163	Medulla	Female
SA175398	900483-015-075	Medulla	Male
SA175399	900483-014-064	Medulla	Male
SA175400	900483-012-042	Medulla	Male
SA175401	900483-016-086	Medulla	Male
SA175402	900483-011-031	Medulla	Male
SA175403	900483-010-020	Medulla	Male
SA175404	900483-009-009	Medulla	Male
SA175405	900483-013-053	Medulla	Male
SA175406	900483-016-170	Midbrain	Female
SA175407	900483-011-115	Midbrain	Female
SA175408	900483-010-104	Midbrain	Female
SA175409	900483-009-093	Midbrain	Female

Showing page 1 of 2 Results: 1 2 Next Showing results 1 to 100 of 175

Collection:

Collection ID:	CO001959
Collection Summary:	Brain tissue samples were collected from 92 weeks old male and female wild type mice on a C57BL/6N 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:	TR001978
Treatment Summary:	Five milligrams of tissue from each brain region were homogenized in 225 µL of -20˚C 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 -20˚C cold, internal standard-containing methyl tertiary-butyl ether (MTBE) was added, and the mixture was vortexed for 10 s and shaken at 4˚C 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 20s to induce phase separation. After centrifugation for 2min 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 30min.

Treatment ID:

TR001978

Treatment Summary:

Five milligrams of tissue from each brain region were homogenized in 225 µL of -20˚C 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 -20˚C cold, internal standard-containing methyl tertiary-butyl ether (MTBE) was added, and the mixture was vortexed for 10 s and shaken at 4˚C 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 20s to induce phase separation. After centrifugation for 2min 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 30min.

Sample Preparation:

Sampleprep ID:	SP001972
Sampleprep Summary:	Five milligrams of tissue from each brain region were homogenized in 225 µL of -20˚C cold, internal standard-containing methanol using a GenoGrinder 2010 (SPEX SamplePrep) for 2min at 1,350 rpm. The homogenate was vortexed for 10s. 750 µL of -20˚C cold, internal standard-containing methyl tertiary-butyl ether (MTBE) was added, and the mixture was vortexed for 10 s and shaken at 4˚C for 5min 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 20s to induce phase separation. After centrifugation for 2min 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 30min.

Sampleprep ID:

SP001972

Sampleprep Summary:

Five milligrams of tissue from each brain region were homogenized in 225 µL of -20˚C cold, internal standard-containing methanol using a GenoGrinder 2010 (SPEX SamplePrep) for 2min at 1,350 rpm. The homogenate was vortexed for 10s. 750 µL of -20˚C cold, internal standard-containing methyl tertiary-butyl ether (MTBE) was added, and the mixture was vortexed for 10 s and shaken at 4˚C for 5min 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 20s to induce phase separation. After centrifugation for 2min 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 30min.

Combined analysis:

Analysis ID	AN003057	AN003058	AN003059	AN003060	AN003061
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:	CH002263
Chromatography Summary:	HILIC positive
Instrument Name:	Thermo Vanquish
Column Name:	Waters XBridge Amide (100 x 4.6mm,3.5um)
Chromatography Type:	HILIC

Chromatography ID:	CH002264
Chromatography Summary:	HILIC negative
Instrument Name:	Thermo Vanquish
Column Name:	Waters XBridge Amide (100 x 4.6mm,3.5um)
Chromatography Type:	HILIC

Chromatography ID:	CH002265
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:	CH002266
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:	CH002267
Chromatography Summary:	GC
Instrument Name:	Agilent 6890N
Column Name:	Restek Rtx-5Sil (30m x 0.25mm,0.25um)
Chromatography Type:	GC

MS:

MS ID:	MS002844
Analysis ID:	AN003057
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:	MS002845
Analysis ID:	AN003058
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:	MS002846
Analysis ID:	AN003059
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:	MS002847
Analysis ID:	AN003060
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:	MS002848
Analysis ID:	AN003061
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