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.

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Study IDST001888
Study TitleA Metabolome Atlas of the Aging Mouse Brain (Study part II)
Study SummaryThe 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
DepartmentGenome Center
LaboratoryWest Coast Metabolomics Center
Last NameDing
First NameJun
Address451 East Health Science Drive, Davis, CA, 95616, USA
Emailjunding@ucdavis.edu
Phone773-326-5420
Submit Date2021-07-25
Raw Data AvailableYes
Raw Data File Type(s)cdf, raw(Thermo)
Analysis Type DetailGC/LC-MS
Release Date2021-08-30
Release Version1
Jun Ding Jun Ding
https://dx.doi.org/10.21228/M8C68D
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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
SA175310900483-015-165Basal ganglia Female
SA175311900483-010-110Basal ganglia Female
SA175312900483-011-121Basal ganglia Female
SA175313900483-016-176Basal ganglia Female
SA175314900483-014-154Basal ganglia Female
SA175315900483-009-099Basal ganglia Female
SA175316900483-013-143Basal ganglia Female
SA175317900483-012-132Basal ganglia Female
SA175318900483-014-066Basal ganglia Male
SA175319900483-011-033Basal ganglia Male
SA175320900483-015-077Basal ganglia Male
SA175321900483-012-044Basal ganglia Male
SA175322900483-016-088Basal ganglia Male
SA175323900483-013-055Basal ganglia Male
SA175324900483-010-022Basal ganglia Male
SA175325900483-009-011Basal ganglia Male
SA175326900483-010-103Cerebellum Female
SA175327900483-011-114Cerebellum Female
SA175328900483-013-136Cerebellum Female
SA175329900483-012-125Cerebellum Female
SA175330900483-014-147Cerebellum Female
SA175331900483-016-169Cerebellum Female
SA175332900483-015-158Cerebellum Female
SA175333900483-009-092Cerebellum Female
SA175334900483-013-048Cerebellum Male
SA175335900483-011-026Cerebellum Male
SA175336900483-009-004Cerebellum Male
SA175337900483-010-015Cerebellum Male
SA175338900483-015-070Cerebellum Male
SA175339900483-016-081Cerebellum Male
SA175340900483-012-037Cerebellum Male
SA175341900483-014-059Cerebellum Male
SA175342900483-009-090Cerebral cortex Female
SA175343900483-012-123Cerebral cortex Female
SA175344900483-011-112Cerebral cortex Female
SA175345900483-014-145Cerebral cortex Female
SA175346900483-016-167Cerebral cortex Female
SA175347900483-015-156Cerebral cortex Female
SA175348900483-013-134Cerebral cortex Female
SA175349900483-010-101Cerebral cortex Female
SA175350900483-016-079Cerebral cortex Male
SA175351900483-010-013Cerebral cortex Male
SA175352900483-013-046Cerebral cortex Male
SA175353900483-012-035Cerebral cortex Male
SA175354900483-014-057Cerebral cortex Male
SA175355900483-009-002Cerebral cortex Male
SA175356900483-011-024Cerebral cortex Male
SA175357900483-015-068Cerebral cortex Male
SA175358900483-013-135Hippocampus Female
SA175359900483-010-102Hippocampus Female
SA175360900483-012-124Hippocampus Female
SA175361900483-009-091Hippocampus Female
SA175362900483-014-146Hippocampus Female
SA175363900483-015-157Hippocampus Female
SA175364900483-011-113Hippocampus Female
SA175365900483-016-168Hippocampus Female
SA175366900483-009-003Hippocampus Male
SA175367900483-013-047Hippocampus Male
SA175368900483-011-025Hippocampus Male
SA175369900483-014-058Hippocampus Male
SA175370900483-012-036Hippocampus Male
SA175371900483-016-080Hippocampus Male
SA175372900483-015-069Hippocampus Male
SA175373900483-010-014Hippocampus Male
SA175374900483-012-128Hypothalamus Female
SA175375900483-015-161Hypothalamus Female
SA175376900483-013-139Hypothalamus Female
SA175377900483-011-117Hypothalamus Female
SA175378900483-010-106Hypothalamus Female
SA175379900483-016-172Hypothalamus Female
SA175380900483-009-095Hypothalamus Female
SA175381900483-014-150Hypothalamus Female
SA175382900483-013-051Hypothalamus Male
SA175383900483-014-062Hypothalamus Male
SA175384900483-009-007Hypothalamus Male
SA175385900483-012-040Hypothalamus Male
SA175386900483-011-029Hypothalamus Male
SA175387900483-015-073Hypothalamus Male
SA175388900483-010-018Hypothalamus Male
SA175389900483-016-084Hypothalamus Male
SA175390900483-013-141Medulla Female
SA175391900483-010-108Medulla Female
SA175392900483-016-174Medulla Female
SA175393900483-011-119Medulla Female
SA175394900483-009-097Medulla Female
SA175395900483-012-130Medulla Female
SA175396900483-014-152Medulla Female
SA175397900483-015-163Medulla Female
SA175398900483-015-075Medulla Male
SA175399900483-014-064Medulla Male
SA175400900483-012-042Medulla Male
SA175401900483-016-086Medulla Male
SA175402900483-011-031Medulla Male
SA175403900483-010-020Medulla Male
SA175404900483-009-009Medulla Male
SA175405900483-013-053Medulla Male
SA175406900483-016-170Midbrain Female
SA175407900483-011-115Midbrain Female
SA175408900483-010-104Midbrain Female
SA175409900483-009-093Midbrain Female
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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.

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.

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 MS (30 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 MS (30 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
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