#METABOLOMICS WORKBENCH huaqisu_20250225_200116 DATATRACK_ID:5677 STUDY_ID:ST003769 ANALYSIS_ID:AN006187 PROJECT_ID:PR002351 VERSION 1 CREATED_ON March 3, 2025, 10:11 pm #PROJECT PR:PROJECT_TITLE Characterization of brain‐derived extracellular vesicle lipids in Alzheimer's PR:PROJECT_TITLE disease PR:PROJECT_TYPE MS lipidomic quantitative analysis PR:PROJECT_SUMMARY Lipid dyshomeostasis is associated with the most common form of dementia, PR:PROJECT_SUMMARY Alzheimer's disease (AD). Substantial progress has been made in identifying PR:PROJECT_SUMMARY positron emission tomography and cerebrospinal fluid biomarkers for AD, but they PR:PROJECT_SUMMARY have limited use as front‐line diagnostic tools. Extracellular vesicles (EVs) PR:PROJECT_SUMMARY are released by all cells and contain a subset of their parental cell PR:PROJECT_SUMMARY composition, including lipids. EVs are released from the brain into the PR:PROJECT_SUMMARY periphery, providing a potential source of tissue and disease specific lipid PR:PROJECT_SUMMARY biomarkers. However, the EV lipidome of the central nervous system is currently PR:PROJECT_SUMMARY unknown and the potential of brain‐derived EVs (BDEVs) to inform on lipid PR:PROJECT_SUMMARY dyshomeostasis in AD remains unclear. The aim of this study was to reveal the PR:PROJECT_SUMMARY lipid composition of BDEVs in human frontal cortex, and to determine whether PR:PROJECT_SUMMARY BDEVs have an altered lipid profile in AD. Using semi‐quantitative mass PR:PROJECT_SUMMARY spectrometry, we describe the BDEV lipidome, covering four lipid categories, 17 PR:PROJECT_SUMMARY lipid classes and 692 lipid molecules. BDEVs were enriched in PR:PROJECT_SUMMARY glycerophosphoserine (PS) lipids, a characteristic of small EVs. Here we further PR:PROJECT_SUMMARY report that BDEVs are enriched in ether‐containing PS lipids, a finding that PR:PROJECT_SUMMARY further establishes ether lipids as a feature of EVs. BDEVs in the AD frontal PR:PROJECT_SUMMARY cortex offered improved detection of dysregulated lipids in AD over global lipid PR:PROJECT_SUMMARY profiling of this brain region. AD BDEVs had significantly altered PR:PROJECT_SUMMARY glycerophospholipid and sphingolipid levels, specifically increased plasmalogen PR:PROJECT_SUMMARY glycerophosphoethanolamine and decreased polyunsaturated fatty acyl containing PR:PROJECT_SUMMARY lipids, and altered amide‐linked acyl chain content in sphingomyelin and PR:PROJECT_SUMMARY ceramide lipids relative to CTL. The most prominent alteration was a two‐fold PR:PROJECT_SUMMARY decrease in lipid species containing anti‐inflammatory/pro‐resolving PR:PROJECT_SUMMARY docosahexaenoic acid. The in‐depth lipidome analysis provided in this study PR:PROJECT_SUMMARY highlights the advantage of EVs over more complex tissues for improved detection PR:PROJECT_SUMMARY of dysregulated lipids that may serve as potential biomarkers in the periphery. PR:INSTITUTE The University of Melbourne PR:DEPARTMENT The Florey Institute of Neuroscience and Mental Health PR:LAST_NAME Su PR:FIRST_NAME Huaqi PR:ADDRESS 30 Royal Parade, Melbourne, VIC, 3150, Australia PR:EMAIL huaqi.su@unimelb.edu.au PR:PHONE +61416373787 #STUDY ST:STUDY_TITLE Characterization of brain‐derived extracellular vesicle lipids in Alzheimer's ST:STUDY_TITLE disease ST:STUDY_SUMMARY Lipid dyshomeostasis is associated with the most common form of dementia, ST:STUDY_SUMMARY Alzheimer's disease (AD). Substantial progress has been made in identifying ST:STUDY_SUMMARY positron emission tomography and cerebrospinal fluid biomarkers for AD, but they ST:STUDY_SUMMARY have limited use as front‐line diagnostic tools. Extracellular vesicles (EVs) ST:STUDY_SUMMARY are released by all cells and contain a subset of their parental cell ST:STUDY_SUMMARY composition, including lipids. EVs are released from the brain into the ST:STUDY_SUMMARY periphery, providing a potential source of tissue and disease specific lipid ST:STUDY_SUMMARY biomarkers. However, the EV lipidome of the central nervous system is currently ST:STUDY_SUMMARY unknown and the potential of brain‐derived EVs (BDEVs) to inform on lipid ST:STUDY_SUMMARY dyshomeostasis in AD remains unclear. The aim of this study was to reveal the ST:STUDY_SUMMARY lipid composition of BDEVs in human frontal cortex, and to determine whether ST:STUDY_SUMMARY BDEVs have an altered lipid profile in AD. Using semi‐quantitative mass ST:STUDY_SUMMARY spectrometry, we describe the BDEV lipidome, covering four lipid categories, 17 ST:STUDY_SUMMARY lipid classes and 692 lipid molecules. BDEVs were enriched in ST:STUDY_SUMMARY glycerophosphoserine (PS) lipids, a characteristic of small EVs. Here we further ST:STUDY_SUMMARY report that BDEVs are enriched in ether‐containing PS lipids, a finding that ST:STUDY_SUMMARY further establishes ether lipids as a feature of EVs. BDEVs in the AD frontal ST:STUDY_SUMMARY cortex offered improved detection of dysregulated lipids in AD over global lipid ST:STUDY_SUMMARY profiling of this brain region. AD BDEVs had significantly altered ST:STUDY_SUMMARY glycerophospholipid and sphingolipid levels, specifically increased plasmalogen ST:STUDY_SUMMARY glycerophosphoethanolamine and decreased polyunsaturated fatty acyl containing ST:STUDY_SUMMARY lipids, and altered amide‐linked acyl chain content in sphingomyelin and ST:STUDY_SUMMARY ceramide lipids relative to CTL. The most prominent alteration was a two‐fold ST:STUDY_SUMMARY decrease in lipid species containing anti‐inflammatory/pro‐resolving ST:STUDY_SUMMARY docosahexaenoic acid. The in‐depth lipidome analysis provided in this study ST:STUDY_SUMMARY highlights the advantage of EVs over more complex tissues for improved detection ST:STUDY_SUMMARY of dysregulated lipids that may serve as potential biomarkers in the periphery. ST:INSTITUTE The University of Melbourne ST:DEPARTMENT The Florey Institute of Neuroscience and Mental Health ST:LAST_NAME Su ST:FIRST_NAME Huaqi ST:ADDRESS 30 Royal Parade, Melbourne, VIC, 3150, Australia ST:EMAIL huaqi.su@unimelb.edu.au ST:PHONE +61416373787 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS 051071CTL 051071CTL_T Sample source:Brain | Diagnosis:CTL RAW_FILE_NAME=CTL01BB1T1-051071--CTL__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL01BB1T1-051071--CTL__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL01BB1T1-051071--CTL__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL01BB1T1-051071--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL01BB1T1-051071--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL01BB1T1-051071--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL01BB1T1-051071--CTL__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 06379AD 06379AD_T Sample source:Brain | Diagnosis:AD RAW_FILE_NAME=AD01BB1T1-06379--AD__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD01BB1T1-06379--AD__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD01BB1T1-06379--AD__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD01BB1T1-06379--AD__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD01BB1T1-06379--AD__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD01BB1T1-06379--AD__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD01BB1T1-06379--AD__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 06972CTL 06972CTL_T Sample source:Brain | Diagnosis:CTL RAW_FILE_NAME=CTL02BB1T1-06972--CTL__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL02BB1T1-06972--CTL__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL02BB1T1-06972--CTL__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL02BB1T1-06972--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL02BB1T1-06972--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL02BB1T1-06972--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL02BB1T1-06972--CTL__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 07022CTL 07022CTL_T Sample source:Brain | Diagnosis:CTL RAW_FILE_NAME=CTL03BB1T1-07022--CTL__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL03BB1T1-07022--CTL__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL03BB1T1-07022--CTL__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL03BB1T1-07022--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL03BB1T1-07022--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL03BB1T1-07022--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL03BB1T1-07022--CTL__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 07284CTL 07284CTL_T Sample source:Brain | Diagnosis:CTL RAW_FILE_NAME=CTL04BB1T1-07284--CTL__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL04BB1T1-07284--CTL__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL04BB1T1-07284--CTL__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL04BB1T1-07284--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL04BB1T1-07284--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL04BB1T1-07284--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL04BB1T1-07284--CTL__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 07635CTL 07635CTL_T Sample source:Brain | Diagnosis:CTL RAW_FILE_NAME=CTL05BB1T1-07635--CTL__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL05BB1T1-07635--CTL__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL05BB1T1-07635--CTL__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL05BB1T1-07635--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL05BB1T1-07635--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL05BB1T1-07635--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL05BB1T1-07635--CTL__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 08007AD 08007AD_T Sample source:Brain | Diagnosis:AD RAW_FILE_NAME=AD02BB1T1-08007--AD__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD02BB1T1-08007--AD__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD02BB1T1-08007--AD__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD02BB1T1-08007--AD__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD02BB1T1-08007--AD__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD02BB1T1-08007--AD__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD02BB1T1-08007--AD__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 08312AD 08312AD_T Sample source:Brain | Diagnosis:AD RAW_FILE_NAME=AD03BB2T1-08312--AD__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD03BB2T1-08312--AD__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD03BB2T1-08312--AD__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD03BB2T1-08312--AD__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD03BB2T1-08312--AD__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD03BB2T1-08312--AD__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD03BB2T1-08312--AD__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 08329AD 08329AD_T Sample source:Brain | Diagnosis:AD RAW_FILE_NAME=AD04BB1T1-08329--AD__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD04BB1T1-08329--AD__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD04BB1T1-08329--AD__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD04BB1T1-08329--AD__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD04BB1T1-08329--AD__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD04BB1T1-08329--AD__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD04BB1T1-08329--AD__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 09006AD 09006AD_T Sample source:Brain | Diagnosis:AD RAW_FILE_NAME=AD05BB1T1-09006--AD__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD05BB1T1-09006--AD__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD05BB1T1-09006--AD__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD05BB1T1-09006--AD__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD05BB1T1-09006--AD__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD05BB1T1-09006--AD__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD05BB1T1-09006--AD__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 09090AD 09090AD_T Sample source:Brain | Diagnosis:AD RAW_FILE_NAME=AD06BB1T1-09090--AD__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD06BB1T1-09090--AD__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD06BB1T1-09090--AD__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD06BB1T1-09090--AD__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD06BB1T1-09090--AD__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD06BB1T1-09090--AD__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD06BB1T1-09090--AD__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 09214AD 09214AD_T Sample source:Brain | Diagnosis:AD RAW_FILE_NAME=AD07BB1T1-09214--AD__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD07BB1T1-09214--AD__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD07BB1T1-09214--AD__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD07BB1T1-09214--AD__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD07BB1T1-09214--AD__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD07BB1T1-09214--AD__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD07BB1T1-09214--AD__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS V11052CTL V11052CTL_T Sample source:Brain | Diagnosis:CTL RAW_FILE_NAME=CTL06BB1T1-V11052--CTL__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL06BB1T1-V11052--CTL__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL06BB1T1-V11052--CTL__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL06BB1T1-V11052--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL06BB1T1-V11052--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL06BB1T1-V11052--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL06BB1T1-V11052--CTL__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS V13052CTL V13052CTL_T Sample source:Brain | Diagnosis:CTL RAW_FILE_NAME=CTL07BB1T1-V13052--CTL__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL07BB1T1-V13052--CTL__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL07BB1T1-V13052--CTL__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL07BB1T1-V13052--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL07BB1T1-V13052--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL07BB1T1-V13052--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL07BB1T1-V13052--CTL__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS V14035CTL V14035CTL_T Sample source:Brain | Diagnosis:CTL RAW_FILE_NAME=CTL08BB1T1-V14035--CTL__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL08BB1T1-V14035--CTL__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL08BB1T1-V14035--CTL__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL08BB1T1-V14035--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL08BB1T1-V14035--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL08BB1T1-V14035--CTL__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL08BB1T1-V14035--CTL__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS V15016AD V15016AD_T Sample source:Brain | Diagnosis:AD RAW_FILE_NAME=AD08BB1T1-V15016--AD__T_12__5X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD08BB1T1-V15016--AD__T_12__5X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD08BB1T1-V15016--AD__T_12__5X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD08BB1T1-V15016--AD__T_12__5X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD08BB1T1-V15016--AD__T_12__5X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD08BB1T1-V15016--AD__T_12__5X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD08BB1T1-V15016--AD__T_12__5X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 051071CTL 051071CTL_F2 Sample source:BDEV | Diagnosis:CTL RAW_FILE_NAME=CTL01CB1T1-051071--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL01CB1T1-051071--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL01CB1T1-051071--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL01CB1T1-051071--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL01CB1T1-051071--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL01CB1T1-051071--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL01CB1T1-051071--CTL__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 06379AD 06379AD_F2 Sample source:BDEV | Diagnosis:AD RAW_FILE_NAME=AD01CB1T1-06379--AD__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD01CB1T1-06379--AD__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD01CB1T1-06379--AD__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD01CB1T1-06379--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD01CB1T1-06379--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD01CB1T1-06379--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD01CB1T1-06379--AD__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 06972CTL 06972CTL_F2 Sample source:BDEV | Diagnosis:CTL RAW_FILE_NAME=CTL02CB1T1-06972--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL02CB1T1-06972--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL02CB1T1-06972--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL02CB1T1-06972--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL02CB1T1-06972--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL02CB1T1-06972--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL02CB1T1-06972--CTL__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 07022CTL 07022CTL_F2 Sample source:BDEV | Diagnosis:CTL RAW_FILE_NAME=CTL03CB1T1-07022--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL03CB1T1-07022--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL03CB1T1-07022--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL03CB1T1-07022--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL03CB1T1-07022--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL03CB1T1-07022--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL03CB1T1-07022--CTL__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 07284CTL 07284CTL_F2 Sample source:BDEV | Diagnosis:CTL RAW_FILE_NAME=CTL04CB1T1-07284--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL04CB1T1-07284--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL04CB1T1-07284--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL04CB1T1-07284--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL04CB1T1-07284--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL04CB1T1-07284--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL04CB1T1-07284--CTL__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 07635CTL 07635CTL_F2 Sample source:BDEV | Diagnosis:CTL RAW_FILE_NAME=CTL05CB1T1-07635--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL05CB1T1-07635--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL05CB1T1-07635--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL05CB1T1-07635--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL05CB1T1-07635--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL05CB1T1-07635--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL05CB1T1-07635--CTL__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 08007AD 08007AD_F2 Sample source:BDEV | Diagnosis:AD RAW_FILE_NAME=AD02CB1T1-08007--AD__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD02CB1T1-08007--AD__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD02CB1T1-08007--AD__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD02CB1T1-08007--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD02CB1T1-08007--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD02CB1T1-08007--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD02CB1T1-08007--AD__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 08312AD 08312AD_F2 Sample source:BDEV | Diagnosis:AD RAW_FILE_NAME=AD03CB2T1-08312--AD__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD03CB2T1-08312--AD__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD03CB2T1-08312--AD__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD03CB2T1-08312--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD03CB2T1-08312--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD03CB2T1-08312--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD03CB2T1-08312--AD__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 08329AD 08329AD_F2 Sample source:BDEV | Diagnosis:AD RAW_FILE_NAME=AD04CB1T1-08329--AD__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD04CB1T1-08329--AD__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD04CB1T1-08329--AD__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD04CB1T1-08329--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD04CB1T1-08329--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD04CB1T1-08329--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD04CB1T1-08329--AD__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 09006AD 09006AD_F2 Sample source:BDEV | Diagnosis:AD RAW_FILE_NAME=AD05CB1T1-09006--AD__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD05CB1T1-09006--AD__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD05CB1T1-09006--AD__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD05CB1T1-09006--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD05CB1T1-09006--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD05CB1T1-09006--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD05CB1T1-09006--AD__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 09090AD 09090AD_F2 Sample source:BDEV | Diagnosis:AD RAW_FILE_NAME=AD06CB1T1-09090--AD__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD06CB1T1-09090--AD__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD06CB1T1-09090--AD__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD06CB1T1-09090--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD06CB1T1-09090--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD06CB1T1-09090--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD06CB1T1-09090--AD__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS 09214AD 09214AD_F2 Sample source:BDEV | Diagnosis:AD RAW_FILE_NAME=AD07CB1T1-09214--AD__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD07CB1T1-09214--AD__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD07CB1T1-09214--AD__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD07CB1T1-09214--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD07CB1T1-09214--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD07CB1T1-09214--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD07CB1T1-09214--AD__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS V11052CTL V11052CTL_F2 Sample source:BDEV | Diagnosis:CTL RAW_FILE_NAME=CTL06CB1T1-V11052--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL06CB1T1-V11052--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL06CB1T1-V11052--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL06CB1T1-V11052--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL06CB1T1-V11052--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL06CB1T1-V11052--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL06CB1T1-V11052--CTL__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS V13052CTL V13052CTL_F2 Sample source:BDEV | Diagnosis:CTL RAW_FILE_NAME=CTL07CB1T1-V13052--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL07CB1T1-V13052--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL07CB1T1-V13052--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL07CB1T1-V13052--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL07CB1T1-V13052--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL07CB1T1-V13052--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL07CB1T1-V13052--CTL__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS V14035CTL V14035CTL_F2 Sample source:BDEV | Diagnosis:CTL RAW_FILE_NAME=CTL08CB1T1-V14035--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=CTL08CB1T1-V14035--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=CTL08CB1T1-V14035--CTL__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=CTL08CB1T1-V14035--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=CTL08CB1T1-V14035--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=CTL08CB1T1-V14035--CTL__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=CTL08CB1T1-V14035--CTL__F2_6__25X_MS_KS_190704_Der_POS_1.RAW SUBJECT_SAMPLE_FACTORS V15016AD V15016AD_F2 Sample source:BDEV | Diagnosis:AD RAW_FILE_NAME=AD08CB1T1-V15016--AD__F2_6__25X_MS_KS_190704_NonDer_POS_1.RAW; RAW_FILE_NAME=AD08CB1T1-V15016--AD__F2_6__25X_MS_KS_190704_NonDer_POS_2.RAW; RAW_FILE_NAME=AD08CB1T1-V15016--AD__F2_6__25X_MS_KS_190704_NonDer_POS_3.RAW; RAW_FILE_NAME=AD08CB1T1-V15016--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_1.RAW; RAW_FILE_NAME=AD08CB1T1-V15016--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_2.RAW; RAW_FILE_NAME=AD08CB1T1-V15016--AD__F2_6__25X_MS_KS_190704_NonDer_NEG_3.RAW; RAW_FILE_NAME=AD08CB1T1-V15016--AD__F2_6__25X_MS_KS_190704_Der_POS_1.RAW #COLLECTION CO:COLLECTION_SUMMARY Fresh frozen human post-mortem frontal cortex tissues of n = 8 AD male subjects CO:COLLECTION_SUMMARY (mean age 74.5 ± SD 7.0 years) and n = 8 gender and age-matched CTL subjects CO:COLLECTION_SUMMARY (mean age 73.5 ± SD 5.9 years) with no evidence of dementia, stored at CO:COLLECTION_SUMMARY −80◦C, were obtained from the Victoria Brain Bank. The average post-mortem CO:COLLECTION_SUMMARY delay before tissue collection was 23.3 ± 17.4 h for AD and 42 ± 16.3 h for CO:COLLECTION_SUMMARY CTL. Frozen frontal cortex tissues (approximately 2 g) were sliced lengthways on CO:COLLECTION_SUMMARY ice to generate 1–2 cm long, 2–3 mm wide tissue sections. Approximately 30 CO:COLLECTION_SUMMARY mg tissue pieces from each sample (“Brain Total”) were collected, weighed CO:COLLECTION_SUMMARY and placed in 19x volume of tissue weight of Dulbecco’s phosphate buffered CO:COLLECTION_SUMMARY saline (DPBS, Thermo Fisher Scientific) solution containing 1x PhosSTOP CO:COLLECTION_SUMMARY phosphatase inhibitor (Sigma Aldrich) / cOmplete protease inhibitor (including CO:COLLECTION_SUMMARY EDTA, Sigma Aldrich) for immunoblot analysis. The remaining cut tissue sections CO:COLLECTION_SUMMARY were weighed and incubated with 50 U/ml collagenase type 3 (#CLS-3, CO:COLLECTION_SUMMARY CAT#LS004182, Worthington) digestion buffer (at ratio of 8μl / mg tissue) in a CO:COLLECTION_SUMMARY shaking water bath (25◦C, a total of 20 min). During incubation, tissue slices CO:COLLECTION_SUMMARY were inverted twice at the 10-min time point, gently pipetted up and down twice CO:COLLECTION_SUMMARY at the 15-min time point and then allowed incubation for a further 5 min, CO:COLLECTION_SUMMARY followed by the addition of ice-cold 10x inhibition buffer, which was made of CO:COLLECTION_SUMMARY 10x phosphatase inhibitor and 10x protease inhibitor in DPBS. The final CO:COLLECTION_SUMMARY concentration of inhibition buffer in solution was 1x. The dissociated tissue in CO:COLLECTION_SUMMARY solution was subjected to a series of centrifugations, including a 300 × g, CO:COLLECTION_SUMMARY 4◦C for 5 min, a 2000 × g, 4◦C for 10 min and a 10,000 × g, 4◦C for 30 CO:COLLECTION_SUMMARY min. Representative 300 × g pellets were collected (‘Brain+C’ for CO:COLLECTION_SUMMARY collagenase treatment) and either placed in 19× volume of tissue weight of DPBS CO:COLLECTION_SUMMARY with 1× phosphatase inhibitor / protease inhibitor solution for protein CO:COLLECTION_SUMMARY quantification and immunoblot analysis or combined with 19× volume of tissue CO:COLLECTION_SUMMARY weight of ice-cold 60% methanol (LCMS grade, EMD Millipore Corporation) CO:COLLECTION_SUMMARY containing 0.01% (w/v) butylated hydroxytoluene (BHT, Sigma Aldrich) for lipid CO:COLLECTION_SUMMARY extraction. The 10,000 × g supernatant was loaded on top of the triple sucrose CO:COLLECTION_SUMMARY density gradient (0.6 M, 1.3M, 2.5 M) as indicated in the method (Vella et al., CO:COLLECTION_SUMMARY 2017) in ultra-clear SW40Ti tubes (Beckman Coulter). The sucrose gradients were CO:COLLECTION_SUMMARY centrifuged at 200,000 x g avg at 4◦C for 173 min using a SW40Ti rotor CO:COLLECTION_SUMMARY (Beckman Coulter). After the spin, the three fractions (F1, F2 and F3, 1.2ml CO:COLLECTION_SUMMARY each) were sequentially collected and refractive index wasmeasured. Each CO:COLLECTION_SUMMARY fraction was subjected to a wash spin in ice-cold DPBS at 128,000 × g avg, at CO:COLLECTION_SUMMARY 4◦C for 80 min using a F37L-8 × 100 rotor (Thermo Fisher Scientific). The CO:COLLECTION_SUMMARY pelleted EVs were resuspended in 150 μl ice-cold DPBS with 1x phosphatase CO:COLLECTION_SUMMARY inhibitor / protease inhibitor solution. CO:COLLECTION_PROTOCOL_FILENAME Su_et_al_2021.pdf CO:SAMPLE_TYPE brain frontal cortex, brain derived extracellular vesicles #TREATMENT TR:TREATMENT_SUMMARY There is no treatment in this study. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Fresh frozen human post-mortem frontal cortex tissues of n = 8 AD male subjects SP:SAMPLEPREP_SUMMARY (mean age 74.5 ± SD 7.0 years) and n = 8 gender and age-matched CTL subjects SP:SAMPLEPREP_SUMMARY (mean age 73.5 ± SD 5.9 years) with no evidence of dementia, stored at SP:SAMPLEPREP_SUMMARY −80◦C, were obtained from the Victoria Brain Bank. The average post-mortem SP:SAMPLEPREP_SUMMARY delay before tissue collection was 23.3 ± 17.4 h for AD and 42 ± 16.3 h for SP:SAMPLEPREP_SUMMARY CTL. Frozen frontal cortex tissues (approximately 2 g) were sliced lengthways on SP:SAMPLEPREP_SUMMARY ice to generate 1–2 cm long, 2–3 mm wide tissue sections. Approximately 30 SP:SAMPLEPREP_SUMMARY mg tissue pieces from each sample (“Brain Total”) were collected, weighed SP:SAMPLEPREP_SUMMARY and placed in 19x volume of tissue weight of Dulbecco’s phosphate buffered SP:SAMPLEPREP_SUMMARY saline (DPBS, Thermo Fisher Scientific) solution containing 1x PhosSTOP SP:SAMPLEPREP_SUMMARY phosphatase inhibitor (Sigma Aldrich) / cOmplete protease inhibitor (including SP:SAMPLEPREP_SUMMARY EDTA, Sigma Aldrich) for immunoblot analysis. The remaining cut tissue sections SP:SAMPLEPREP_SUMMARY were weighed and incubated with 50 U/ml collagenase type 3 (#CLS-3, SP:SAMPLEPREP_SUMMARY CAT#LS004182, Worthington) digestion buffer (at ratio of 8μl / mg tissue) in a SP:SAMPLEPREP_SUMMARY shaking water bath (25◦C, a total of 20 min). During incubation, tissue slices SP:SAMPLEPREP_SUMMARY were inverted twice at the 10-min time point, gently pipetted up and down twice SP:SAMPLEPREP_SUMMARY at the 15-min time point and then allowed incubation for a further 5 min, SP:SAMPLEPREP_SUMMARY followed by the addition of ice-cold 10x inhibition buffer, which was made of SP:SAMPLEPREP_SUMMARY 10x phosphatase inhibitor and 10x protease inhibitor in DPBS. The final SP:SAMPLEPREP_SUMMARY concentration of inhibition buffer in solution was 1x. The dissociated tissue in SP:SAMPLEPREP_SUMMARY solution was subjected to a series of centrifugations, including a 300 × g, SP:SAMPLEPREP_SUMMARY 4◦C for 5 min, a 2000 × g, 4◦C for 10 min and a 10,000 × g, 4◦C for 30 SP:SAMPLEPREP_SUMMARY min. Representative 300 × g pellets were collected (‘Brain+C’ for SP:SAMPLEPREP_SUMMARY collagenase treatment) and either placed in 19× volume of tissue weight of DPBS SP:SAMPLEPREP_SUMMARY with 1× phosphatase inhibitor / protease inhibitor solution for protein SP:SAMPLEPREP_SUMMARY quantification and immunoblot analysis or combined with 19× volume of tissue SP:SAMPLEPREP_SUMMARY weight of ice-cold 60% methanol (LCMS grade, EMD Millipore Corporation) SP:SAMPLEPREP_SUMMARY containing 0.01% (w/v) butylated hydroxytoluene (BHT, Sigma Aldrich) for lipid SP:SAMPLEPREP_SUMMARY extraction. The 10,000 × g supernatant was loaded on top of the triple sucrose SP:SAMPLEPREP_SUMMARY density gradient (0.6 M, 1.3M, 2.5 M) as indicated in the method (Vella et al., SP:SAMPLEPREP_SUMMARY 2017) in ultra-clear SW40Ti tubes (Beckman Coulter). The sucrose gradients were SP:SAMPLEPREP_SUMMARY centrifuged at 200,000 x g avg at 4◦C for 173 min using a SW40Ti rotor SP:SAMPLEPREP_SUMMARY (Beckman Coulter). After the spin, the three fractions (F1, F2 and F3, 1.2ml SP:SAMPLEPREP_SUMMARY each) were sequentially collected and refractive index wasmeasured. Each SP:SAMPLEPREP_SUMMARY fraction was subjected to a wash spin in ice-cold DPBS at 128,000 × g avg, at SP:SAMPLEPREP_SUMMARY 4◦C for 80 min using a F37L-8 × 100 rotor (Thermo Fisher Scientific). The SP:SAMPLEPREP_SUMMARY pelleted EVs were resuspended in 150 μl ice-cold DPBS with 1x phosphatase SP:SAMPLEPREP_SUMMARY inhibitor / protease inhibitor solution. SP:SAMPLEPREP_PROTOCOL_FILENAME Su_et_al_2021.pdf SP:EXTRACTION_METHOD The “total brain with collagenase” tissue pellets in ice-cold 60%methanol SP:EXTRACTION_METHOD containing 0.01% (w/v) BHTwere homogenised using a cell disrupter as described SP:EXTRACTION_METHOD above. 100 μl of the homogenates were combined with 100 μl of 60%methanol SP:EXTRACTION_METHOD containing 0.01% (w/v) BHT. 80 μl of the F2BDEVsuspensionswere combinedwith 20 SP:EXTRACTION_METHOD μl of ice-coldmethanolwith0.1%(w/v)BHTand100μl of ice-cold methanol to make a SP:EXTRACTION_METHOD final volume of 200 μl 60%methanol containing 0.01% (w/v) BHT. All samples were SP:EXTRACTION_METHOD sonicated in an ice-cold water bath sonicator (20min) prior to lipid SP:EXTRACTION_METHOD extraction.Monophasic lipid extraction followed the method previously reported SP:EXTRACTION_METHOD by Lydic et al. (Lydic et al., 2015) with modification as described below. 120 SP:EXTRACTION_METHOD μl of MilliQwater, 420 μl of methanol with 0.01% (w/v) BHT, and 270 μl of SP:EXTRACTION_METHOD chloroform were added to all samples. For every 10 μg protein present in the SP:EXTRACTION_METHOD samples, 1 μl of a customised isotope labelled internal standard lipid mixture SP:EXTRACTION_METHOD and 1 μl of a d5-TG Internal Standard Mixture I (Avanti Polar Lipids, SP:EXTRACTION_METHOD Alabaster, AL, USA) were added. The customised isotope labelled internal SP:EXTRACTION_METHOD standard mixture was comprised of 14 deuterated lipid standards (Avanti Polar SP:EXTRACTION_METHOD Lipids, Alabaster, AL, USA): 15:0-18:1(d7) PC (250 μM), 15:0-18:1(d7) PE (240 SP:EXTRACTION_METHOD μM), 15:0-18:1(d7) PS (250 μM), 15:0-18:1(d7) PG (20 μM), 15:0-18:1(d7) PI SP:EXTRACTION_METHOD (220 μM), 15:0-18:1(d7) PA (180 μM), 18:1(d7) LPC (45 μM), 18:1(d7) LPE (10 SP:EXTRACTION_METHOD μM), 18:1(d7) Chol Ester (10 μM), 18:1(d7) MG (10 μM), 15:0-18:1(d7) DG (17 SP:EXTRACTION_METHOD μM), 18:1(d9) SM (80 μM), d18:1(d7)-15:0 Cer (40 μM) and Cholesterol(d7) (20 SP:EXTRACTION_METHOD μM). The d5-TG Internal StandardMixture I contained 20:5-22:6-20:5 (d5) TG SP:EXTRACTION_METHOD (4.03 μM), 14:0-16:1-14:0 (d5) TG(3.99 μM), 15:0-18:1-15:0 (d5) TG(3.97 μM), SP:EXTRACTION_METHOD 16:0-18:0-16:0 (d5) TG(4.05 μM), 17:0-17:1-17:0 (d5) TG(4.14 μM), 19:0-12:0- SP:EXTRACTION_METHOD 19:0 (d5) TG (4.01 μM), 20:0-20:1-20:0 (d5) TG (3.81 μM), 20:2-18:3-20:2 (d5) SP:EXTRACTION_METHOD TG (3.96 μM), 20:4-18:2-20:4 (d5) TG (3.90 μM). The 15:0-18:1-15:0 (d5) TG was SP:EXTRACTION_METHOD used for semi-quantification of endogenous TG lipids. Samples were vortexed SP:EXTRACTION_METHOD thoroughly and incubated with 1,000 rpmshaking at room temperature for 30min, SP:EXTRACTION_METHOD followed by centrifugation at 14,000 rpmat roomtemperature for 15 min. SP:EXTRACTION_METHOD Supernatants containing lipids were transferred to new tubes. The remaining SP:EXTRACTION_METHOD pellets were re-extracted with 100 μL of MilliQ water and 400 μl of SP:EXTRACTION_METHOD chloroform:methanol (1:2, v:v) containing 0.01% (w/v) butylated hydroxytoluene SP:EXTRACTION_METHOD (BHT) following incubation and centrifugation as described above. The SP:EXTRACTION_METHOD supernatants from the repetitive extractions were collected and pooled, dried by SP:EXTRACTION_METHOD evaporation under vacuum using a GeneVac miVac sample concentrator (SP SP:EXTRACTION_METHOD Scientific, Warminster, PA, USA) and then reconstituted in SP:EXTRACTION_METHOD isopropanol:methanol:chloroform (4:2:1, v:v:v, containing 0.01% BHT) at a final SP:EXTRACTION_METHOD concentration of 4 μl lipid extract per μg protein. SP:SAMPLE_DERIVATIZATION Derivatization of aminophospholipids (i.e., PE and PS) and SP:SAMPLE_DERIVATIZATION plasmalogen-containing lipids followed the method previously reported by Ryan SP:SAMPLE_DERIVATIZATION and Reid (2016). Prior to derivatization, a 2.5 mM stock solution of SP:SAMPLE_DERIVATIZATION triethyamine (TEA) in chloroform was freshly prepared by adding 3.4 μl TEA to SP:SAMPLE_DERIVATIZATION 10 ml of chloroform. A 2.5 mM stock solution of S,S’- SP:SAMPLE_DERIVATIZATION dimethylthiobutanoylhydroxysuccinimide ester iodide (13C1-DMBNHS) was freshly SP:SAMPLE_DERIVATIZATION prepared by dissolving 4.87 mg 13C1- DMBNHS in 5 ml of dimethylformamide (DMF). SP:SAMPLE_DERIVATIZATION A stock solution of 3.94 mM iodine was freshly prepared by dissolving 10 mg SP:SAMPLE_DERIVATIZATION iodine in 10 ml chloroform. A stock solution of 90 mM ammonium bicarbonate was SP:SAMPLE_DERIVATIZATION freshly prepared by dissolving 35.6 mg ammonium bicarbonate in 5 ml of HPLC SP:SAMPLE_DERIVATIZATION methanol. A solution of 2:1 (v:v) chloroform:methanol containing 266 μM iodine SP:SAMPLE_DERIVATIZATION and 2mMammoniumbicarbonatewas prepared by adding 160 μl of 3.94mMiodine in SP:SAMPLE_DERIVATIZATION chloroformto 1.44ml chloroform, and 53.3 μl of 90mMammonium bicarbonate SP:SAMPLE_DERIVATIZATION inmethanol to 746.7ml methanol, then combined and placed in an ice bath. Due to SP:SAMPLE_DERIVATIZATION the limitations in sample amounts, no replicate derivatization reactions were SP:SAMPLE_DERIVATIZATION performed. 4 μl of brain tissue or BDEV lipid extracts were aliquoted to SP:SAMPLE_DERIVATIZATION individual wells of a Whatman Multi-Chem 96-well plate (Sigma Aldrich, St. SP:SAMPLE_DERIVATIZATION Louis, MO, USA). The solvent was evaporated under vacuum with a GeneVac miVac SP:SAMPLE_DERIVATIZATION sample concentrator. 40 μl of a solution of 39:1.1:1 (v:v:v) chloroform:2.5 mM SP:SAMPLE_DERIVATIZATION TEA:2.5 mM 13C1-DMBNHS reagent was added to each dried lipid extract and the SP:SAMPLE_DERIVATIZATION 96-well plate was sealed with Teflon Ultra Thin Sealing Tape. Samples were then SP:SAMPLE_DERIVATIZATION incubated at room temperature with gentle shaking for 30 min. After incubation, SP:SAMPLE_DERIVATIZATION the solvents were evaporated under vacuum with a GeneVac miVac sample SP:SAMPLE_DERIVATIZATION concentrator and samples were chilled on ice for 10 min prior to addition of 40 SP:SAMPLE_DERIVATIZATION μl of the 2:1 (v:v) chloroform:methanol containing 266 μMiodine and 2 SP:SAMPLE_DERIVATIZATION mMammonium bicarbonate. Reactions were mixed by careful pipetting and the plate SP:SAMPLE_DERIVATIZATION was sealed with aluminiumfoil and then placed on ice for 5min before solvents SP:SAMPLE_DERIVATIZATION were completely removed by evaporation under vacuum with a GeneVac miVac sample SP:SAMPLE_DERIVATIZATION concentrator. The dried lipid extracts were washed three times with 40 μl of 10 SP:SAMPLE_DERIVATIZATION mM aqueous ammonium. Remaining traces of water were then removed by evaporation SP:SAMPLE_DERIVATIZATION under vacuum with a GeneVac miVac sample concentrator. The derivatized brain SP:SAMPLE_DERIVATIZATION tissue lipid extracts and BDEV lipid extracts were then resuspended in 50 μl SP:SAMPLE_DERIVATIZATION and 25 μl of isopropanol:methanol:chloroform(4:2:1, v:v:v) containing SP:SAMPLE_DERIVATIZATION 20mMammoniumformate respectively. The 96-well plate was then sealed with Teflon SP:SAMPLE_DERIVATIZATION Ultra Thin Sealing Tape prior to mass spectrometry analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Direct infusion lipidomic analyses were conducted. For underivatized samples, 4 CH:CHROMATOGRAPHY_SUMMARY μl of brain tissue or BDEV lipid extracts were aliquoted in triplicate to CH:CHROMATOGRAPHY_SUMMARY individual wells of a twin-tec 96-well plate (Eppendorf,Hamburg,Germany). The CH:CHROMATOGRAPHY_SUMMARY brain tissue lipid extracts andBDEVlipid extractswere dried and then resuspended CH:CHROMATOGRAPHY_SUMMARY in 50 μl (brain tissue) and 25 μl (BDEV) of CH:CHROMATOGRAPHY_SUMMARY isopropanol:methanol:chloroform(4:2:1, v:v:v) containing 20mMammonium formate CH:CHROMATOGRAPHY_SUMMARY respectively. The 96-well plate was then sealed with Teflon Ultra Thin Sealing CH:CHROMATOGRAPHY_SUMMARY Tape prior to mass spectrometry analysis. 10 μl of each underivatized or CH:CHROMATOGRAPHY_SUMMARY derivatized lipid sample was aspirated and introduced via nano-ESI to an CH:CHROMATOGRAPHY_SUMMARY Orbitrap Fusion Lumos mass spectrometer (ThermoFisher Scientific, San Jose, CA, CH:CHROMATOGRAPHY_SUMMARY USA) using anAdvion TriversaNanomate (Advion, Ithaca,NY, USA) operating with a CH:CHROMATOGRAPHY_SUMMARY spray voltage of 1.1 kV and a gas pressure of 0.3 psi in both positive and CH:CHROMATOGRAPHY_SUMMARY negative ionizationmodes. For MS analysis, the RF lens was set at 10%. Full scan CH:CHROMATOGRAPHY_SUMMARY mass spectra were acquired at a mass resolving power of 500,000 (at 200 m/z) CH:CHROMATOGRAPHY_SUMMARY across a m/z range of 350 – 1600 using quadrupole isolation, with an automatic CH:CHROMATOGRAPHY_SUMMARY gain control (AGC) target of 5e5. Themaximum injection time was set at 50 ms. CH:CHROMATOGRAPHY_SUMMARY Spectra were acquired and averaged for 3 min. Following initial CH:CHROMATOGRAPHY_SUMMARY ‘sum-composition’ lipid assignments by database analysis (see below), CH:CHROMATOGRAPHY_SUMMARY ‘targeted’ higher-energy collision induced dissociation (HCD-MS/MS) product CH:CHROMATOGRAPHY_SUMMARY ion spectra were acquired on selected precursor ions at amass resolving power of CH:CHROMATOGRAPHY_SUMMARY 120,000 and default activation times in positive ionizationmode using the CH:CHROMATOGRAPHY_SUMMARY underivatized lipid extracts to confirm the identities of lipid head groups, or CH:CHROMATOGRAPHY_SUMMARY in negative ionization mode using underivatized lipid extracts for fatty acid CH:CHROMATOGRAPHY_SUMMARY chain identification. HCD-MS/MS collision energies were individually optimized CH:CHROMATOGRAPHY_SUMMARY for each lipid class of interest using commercially available lipid standards CH:CHROMATOGRAPHY_SUMMARY whenever possible. CH:CHROMATOGRAPHY_TYPE None (Direct infusion) CH:INSTRUMENT_NAME none CH:COLUMN_NAME none CH:SOLVENT_A none CH:SOLVENT_B none CH:FLOW_GRADIENT none CH:FLOW_RATE none CH:COLUMN_TEMPERATURE none #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE Su_et_al_2021.pdf #MS MS:INSTRUMENT_NAME Thermo Orbitrap Fusion Lumos Tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Non derivatized lipids were measured in positive mode in triplicate. ‘Sum MS:MS_COMMENTS composition’ level lipid identifications were achieved using a developmental MS:MS_COMMENTS version of LipidSearch software 5.0α (Mitsui Knowledge Industry, Tokyo, Japan) MS:MS_COMMENTS by automated peak peaking and searching against a user-defined custom database MS:MS_COMMENTS of lipid species (including the deuterated internal standard lipid species and MS:MS_COMMENTS allowing for the mass shifts introduced by 13C1-DMBNHS and iodine/methanol MS:MS_COMMENTS derivatization). The parent tolerance was set at 3.0 ppm, a parent ion intensity MS:MS_COMMENTS threshold three times that of the experimentally observed instrument noise MS:MS_COMMENTS intensity, and a max isotope number of 1 (i.e., matching based on the MS:MS_COMMENTS monoisotopic ion and the M+1 isotope), a correlation threshold (%) of 0.3 and an MS:MS_COMMENTS isotope threshold (%) of 0.1. The lipid nomenclature used here follows that MS:MS_COMMENTS defined by the LIPID MAPS consortium (Fahy et al., 2005). Semi-quantification of MS:MS_COMMENTS the abundances of identified lipid species was performed using an in-house R MS:MS_COMMENTS script, by comparing the identified lipid ion peak areas to the peak areas of MS:MS_COMMENTS the internal standard for each lipid class or subclass, followed by MS:MS_COMMENTS normalization against the total protein amount in the samples. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS pmol/µg protein MS_METABOLITE_DATA_START Samples 051071CTL_T 06972CTL_T 07022CTL_T 07284CTL_T 07635CTL_T V13052CTL_T V14035CTL_T V11052CTL_T 06379AD_T 08007AD_T 08312AD_T 08329AD_T 09006AD_T 09090AD_T 09214AD_T V15016AD_T 051071CTL_F2 06972CTL_F2 07022CTL_F2 07284CTL_F2 07635CTL_F2 V11052CTL_F2 V13052CTL_F2 V14035CTL_F2 06379AD_F2 08007AD_F2 08312AD_F2 08329AD_F2 09006AD_F2 09090AD_F2 09214AD_F2 V15016AD_F2 Factors Sample source:Brain | Diagnosis:CTL Sample source:Brain | Diagnosis:CTL Sample source:Brain | Diagnosis:CTL Sample source:Brain | Diagnosis:CTL Sample source:Brain | Diagnosis:CTL Sample source:Brain | Diagnosis:CTL Sample source:Brain | Diagnosis:CTL Sample source:Brain | Diagnosis:CTL Sample source:Brain | Diagnosis:AD Sample source:Brain | Diagnosis:AD Sample source:Brain | Diagnosis:AD Sample source:Brain | Diagnosis:AD Sample source:Brain | Diagnosis:AD Sample source:Brain | Diagnosis:AD Sample source:Brain | Diagnosis:AD Sample source:Brain | Diagnosis:AD Sample source:BDEV | Diagnosis:CTL Sample source:BDEV | Diagnosis:CTL Sample source:BDEV | Diagnosis:CTL Sample source:BDEV | Diagnosis:CTL Sample source:BDEV | Diagnosis:CTL Sample source:BDEV | Diagnosis:CTL Sample source:BDEV | Diagnosis:CTL Sample source:BDEV | Diagnosis:CTL Sample source:BDEV | Diagnosis:AD Sample source:BDEV | Diagnosis:AD Sample source:BDEV | Diagnosis:AD Sample source:BDEV | Diagnosis:AD Sample source:BDEV | Diagnosis:AD Sample source:BDEV | Diagnosis:AD Sample source:BDEV | Diagnosis:AD Sample source:BDEV | Diagnosis:AD CE(16:0) 0.04 0.204 0.042 0.012 0.032 0.017 0.026 0.022 0.013 0.044 0.074 0.268 0.259 0.05 0.833 0.037 0.084 0.015 0.213 0.025 0.037 0.061 0.071 0.123 0.042 0.142 0.231 0.127 0.113 0.06 0.295 0.113 CE(16:1) 0.108 0.332 0.039 0.004 0.019 0.033 0.018 0.004 0.003 0.035 0.051 0.443 0.701 0.02 0.572 0.007 0.196 0.008 0.286 0.011 0.029 0.036 0.01 0.083 0.027 0.154 0.399 0.059 0.04 0.017 0.132 0.042 CE(18:1) 0.213 0.573 0.334 0.078 0.14 0.101 0.151 0.089 0.092 0.165 0.27 0.789 0.585 0.219 1.987 0.22 0.522 0.178 0.816 0.132 0.191 0.348 0.29 0.359 0.255 0.594 0.646 0.536 0.501 0.277 0.99 0.433 CE(18:2) 0.087 0.359 0.424 0.096 0.311 0.117 0.152 0.249 0.033 0.131 0.215 0.441 0.342 0.077 0.611 0.224 0.395 0.308 0.629 0.186 0.366 0.571 0.782 0.724 0.183 0.414 0.538 0.62 0.844 0.123 1.174 0.433 CE(20:4) 0.206 0.168 0.601 0.144 0.158 0.166 0.094 0.261 0.144 0.174 0.363 0.618 0.208 0.331 0.465 0.618 0.333 0.226 0.426 0.251 0.137 0.305 0.331 0.443 0.13 0.18 0.347 0.324 0.323 0.249 0.414 0.366 CE(22:6) 0.01 0.018 0.076 0.014 0.01 0.027 0.009 0.012 0.013 0.014 0.028 0.073 0.02 0.037 0.048 0.059 0.029 0.044 0.08 0.049 0.007 0.078 0.032 0.053 0.01 0 0.026 0.03 0.021 0.033 0.035 0.018 Cer(d24:0) 0.023 0.118 0.041 0.003 0.01 0.116 0.013 0.031 0.017 0.008 0.129 0.198 0.011 0.034 0.111 0.017 0.028 0.016 0.068 0.968 0.016 0.023 0.009 0.022 0.037 13.632 0.033 0.042 0.043 0.027 0.016 0.022 Cer(d30:1) 0.012 0.107 0.012 0.007 0.007 0.018 0.009 0.046 0.006 0.003 0.006 0.009 0.01 0.003 0.032 0.004 0.012 0.028 0.045 0.006 0.004 0 0.006 0.009 0.004 0.107 0.016 0.011 0.066 0.056 0.007 0.012 Cer(d31:3) 0.403 1.064 0.079 0.022 0.024 1.975 0.18 0.455 0.099 0.02 0.022 0.102 0.074 0.026 1.211 0.062 0.081 0.633 0.533 0.027 0.027 0.076 0.018 0.03 0.106 0.926 0.019 0.113 1.041 0.871 0.115 0.102 Cer(d32:0) 0.444 1.043 0.013 0.017 0.01 2.5 0.067 0.36 0.046 0.009 0.014 0.025 0.022 0.054 1.368 0.014 0.018 0.429 0.461 0.01 0.006 0.023 0.007 0.021 0.006 0.874 0.019 0.011 0.918 0.817 0.004 0.011 Cer(d34:0) 0.329 0.868 0.009 0.008 0.004 2.131 0.054 0.288 0.03 0.006 0.01 0.04 0.024 0.031 1.055 0.017 0.021 0.327 0.363 0.008 0.011 0.023 0.015 0.045 0.017 0.754 0.053 0.007 0.743 0.663 0.01 0.028 Cer(d34:1) 0.325 0.721 0.353 0.196 0.222 0.377 0.193 0.415 0.266 0.206 0.23 0.387 0.172 0.401 0.403 0.542 0.442 0.579 0.935 0.379 0.372 0.548 0.457 0.568 0.362 1.101 0.665 0.368 0.782 0.853 0.325 0.615 Cer(d36:0) 0.121 0.386 0.065 0.033 0.021 0.639 0.04 0.127 0.064 0.029 0.066 0.155 0.062 0.067 0.374 0.132 0.062 0.139 0.237 0.033 0.034 0.056 0.073 0.089 0.062 0.326 0.143 0.071 0.279 0.294 0.044 0.089 Cer(d36:1) 3.932 4.631 7.302 4.013 3.137 5.238 3.238 4.386 4.716 4.005 5.683 7.339 3.388 7.107 5.628 9.538 4.268 5.755 8.077 5.687 3.922 6.216 7.242 5.691 3.74 5.464 7.697 6.336 4.173 7.157 4.803 5.035 Cer(d36:2) 1.635 1.669 1.754 1.416 1.241 1.707 1.258 1.754 1.53 1.773 2.045 2.401 1.208 2.906 2.136 2.955 1.565 1.975 2.163 2.105 1.116 1.679 2.606 1.555 0.947 2.008 2.537 2.067 1.3 2.28 1.52 1.5 Cer(d37:1) 0.003 0.054 0.023 0.011 0.006 0.011 0.006 0.004 0.013 0.012 0.021 0.047 0.029 0.016 0.029 0.03 0.006 0.003 0.026 0.014 0.009 0.017 0.02 0.014 0.015 0.109 0.051 0.017 0.007 0.02 0.011 0.017 Cer(d38:1) 0.898 1.244 1.901 0.891 0.98 1.61 1.024 1.054 1.337 1.269 1.189 1.809 1.048 1.887 1.807 2.534 1.137 1.431 2.181 1.48 1.184 1.969 1.88 1.601 1.058 1.55 1.719 1.587 1.306 1.945 1.311 1.251 Cer(d38:2) 0.113 0.151 0.125 0.143 0.137 0.131 0.11 0.16 0.109 0.187 0.178 0.157 0.104 0.314 0.175 0.231 0.138 0.162 0.215 0.203 0.105 0.175 0.271 0.131 0.064 0.18 0.286 0.092 0.084 0.241 0.091 0.109 Cer(d40:1) 0.061 0.217 0.271 0.119 0.054 0.101 0.042 0.068 0.067 0.062 0.238 0.265 0.147 0.168 0.132 0.164 0.15 0.115 0.333 0.11 0.157 0.231 0.165 0.282 0.278 0.498 0.481 0.068 0.207 0.195 0.175 0.321 Cer(d40:2) 0.021 0.087 0.107 0.041 0.018 0.035 0.012 0.024 0.015 0.018 0.092 0.089 0.044 0.059 0.03 0.04 0.047 0.042 0.094 0.03 0.052 0.066 0.053 0.087 0.093 0.221 0.239 0.025 0.061 0.042 0.042 0.154 Cer(d41:1) 0.02 0.088 0.25 0.072 0.012 0.055 0.013 0.01 0.021 0.019 0.204 0.16 0.098 0.087 0.03 0.097 0.028 0.014 0.058 0.014 0.026 0.042 0.034 0.06 0.065 0.078 0.105 0.038 0.031 0.026 0.049 0.061 Cer(d41:2) 0.017 0.085 0.203 0.066 0.019 0.048 0.01 0.011 0.016 0.018 0.169 0.132 0.06 0.092 0.018 0.074 0.023 0.01 0.038 0.019 0.033 0.031 0.027 0.033 0.058 0.035 0.072 0.05 0.03 0.024 0.046 0.068 Cer(d42:1) 0.06 0.204 0.639 0.16 0.026 0.146 0.043 0.017 0.075 0.041 0.331 0.346 0.212 0.21 0.058 0.241 0.151 0.07 0.354 0.092 0.152 0.292 0.176 0.466 0.522 0.691 0.649 0.08 0.244 0.115 0.218 0.515 Cer(d42:2) 1.167 3.053 8.757 2.056 0.556 2.984 0.594 0.549 1.036 0.666 3.842 4.047 2.496 2.298 0.615 3.137 0.764 0.775 2.207 0.848 0.845 1.192 1.117 1.317 1.29 1.71 1.721 1.338 1.053 0.597 0.958 1.664 Cer(d42:3) 0.058 0.158 0.367 0.114 0.05 0.123 0.039 0.029 0.095 0.069 0.298 0.328 0.125 0.187 0.087 0.208 0.071 0.077 0.194 0.056 0.076 0.086 0.113 0.134 0.179 0.202 0.287 0.093 0.1 0.053 0.142 0.216 Cer(d44:2) 0.035 0.118 0.363 0.107 0.02 0.07 0.02 0.005 0.033 0.024 0.16 0.18 0.1 0.091 0.009 0.146 0.015 0 0.054 0.01 0.012 0.013 0.027 0.024 0.02 0.025 0.05 0.017 0.008 0.008 0.005 0.02 Hex1Cer(d33:1) 0.333 0.268 0.239 0.179 0.207 0.282 0.184 0.257 0.243 0.208 0.194 0.228 0.221 0.218 0.264 0.236 0.234 0.238 0.29 0.187 0.202 0.199 0.163 0.24 0.29 0.321 0.279 0.352 0.289 0.231 0.274 0.296 Hex1Cer(d35:2) 1.102 0.962 0.957 0.65 0.695 0.998 0.673 0.81 0.903 0.653 0.704 0.758 0.87 0.93 1.059 0.944 0.817 0.833 1.093 0.65 0.78 0.768 0.611 0.786 1.093 1.187 0.86 1.138 0.976 0.813 0.812 0.992 Hex1Cer(d36:1) 1.193 2.912 7.05 0.868 0.513 3.536 0.584 0.393 0.641 0.531 1.898 3.707 2.129 1.549 0.634 3.121 0.243 0.162 1.604 0.203 0.416 0.485 0.333 0.375 0.213 0.793 0.391 1.642 0.468 0.157 0.475 0.715 Hex1Cer(d36:2) 0.129 0.26 0.584 0.061 0.034 0.31 0.025 0.02 0.045 0.037 0.216 0.328 0.194 0.183 0.043 0.336 0.025 0 0.09 0.008 0.014 0.017 0.01 0.018 0.017 0.125 0.028 0.078 0.066 0.009 0.027 0.038 Hex1Cer(d38:1) 0.105 0.401 0.675 0.119 0.049 0.336 0.062 0.097 0.07 0.054 0.258 0.493 0.244 0.247 0.132 0.306 0.03 0 0.118 0.019 0.024 0.04 0.017 0.034 0.029 0.558 0.102 0.128 0.031 0.012 0.05 0.071 Hex1Cer(d38:2) 10.394 8.551 13.921 5.011 6.327 9.314 4.924 7.685 8.113 6.499 8.34 8.302 8.593 9.486 6.229 10.933 8.491 7.967 9.798 6.125 6.956 5.644 5.65 9.024 10.003 7.937 7.952 14.121 10.215 8.255 6.527 10.907 Hex1Cer(d40:1) 0.284 1.006 1.45 0.337 0.091 0.768 0.132 0.105 0.195 0.094 0.716 1.164 0.694 0.786 0.18 0.83 0.037 0.019 0.223 0.029 0.042 0.071 0.038 0.062 0.081 0.51 0.123 0.387 0.094 0.034 0.092 0.203 Hex1Cer(d40:2) 0.228 0.549 0.631 0.144 0.055 0.388 0.059 0.041 0.103 0.063 0.344 0.561 0.319 0.407 0.099 0.426 0.016 0.01 0.141 0.013 0.027 0.022 0.013 0.047 0.028 0.408 0.091 0.163 0.028 0.02 0.018 0.062 Hex1Cer(d41:1) 0.556 1.451 2.209 0.534 0.172 1.304 0.265 0.14 0.396 0.229 1.15 1.814 1.066 1.383 0.494 1.576 0.066 0.037 0.438 0.061 0.077 0.126 0.106 0.096 0.134 0.335 0.152 0.692 0.187 0.109 0.142 0.319 Hex1Cer(d41:2) 0.362 1.064 1.391 0.317 0.155 0.788 0.139 0.087 0.222 0.137 0.76 1.139 0.616 0.874 0.214 0.819 0.026 0.016 0.251 0.04 0.058 0.071 0.056 0.045 0.048 0.15 0.097 0.414 0.065 0.041 0.064 0.147 Hex1Cer(d42:1) 2.078 4.2 8.526 1.74 0.655 4.357 0.971 0.574 1.576 1.008 3.204 5.879 3.615 4.67 1.312 5.555 0.484 0.26 1.714 0.411 0.507 0.751 0.677 0.667 0.869 1.023 0.696 2.942 0.894 0.517 0.877 1.63 Hex1Cer(d42:2) 12.045 30.767 56.601 9.931 4.719 31.58 5.497 4.284 7.772 5.235 18.523 36.436 21.174 23.887 6.399 28.346 2.433 2.252 11.558 2.551 3.377 4.442 3.626 4.246 3.596 4.753 3.376 15.767 4.796 2.666 4.131 7.424 Hex1Cer(d42:3) 1.379 2.782 3.975 0.777 0.531 2.845 0.508 0.471 0.797 0.588 1.984 2.988 1.792 2.626 0.874 2.373 0.127 0.127 1.002 0.152 0.245 0.265 0.24 0.273 0.212 0.551 0.593 1.209 0.291 0.174 0.336 0.545 Hex1Cer(d43:1) 0.821 1.53 2.751 0.634 0.233 1.371 0.369 0.154 0.654 0.464 1.108 2.259 1.283 1.857 0.523 2.319 0.076 0.037 0.524 0.063 0.113 0.145 0.15 0.132 0.205 0.278 0.157 0.916 0.181 0.08 0.198 0.495 Hex1Cer(d44:1) 0.167 0.241 0.561 0.105 0.028 0.193 0.055 0.016 0.121 0.079 0.14 0.5 0.244 0.332 0.054 0.496 0.012 0 0.051 0.006 0.004 0 0.007 0.004 0.011 0.062 0.006 0.127 0.012 0.009 0 0.07 Hex1Cer(d44:2) 3.037 5.76 9.128 2.003 0.967 4.708 1.303 0.697 2.14 1.579 3.262 7.972 4.425 5.457 1.801 6.649 0.466 0.288 2.152 0.472 0.579 0.688 0.714 0.67 0.803 1.1 0.622 3.433 0.85 0.539 0.844 1.724 Hex1Cer(d44:3) 0.533 0.917 1.184 0.304 0.163 0.775 0.189 0.097 0.365 0.259 0.618 1.31 0.703 1.158 0.37 0.896 0.027 0.024 0.201 0.029 0.04 0.046 0.046 0.037 0.05 0.089 0.088 0.414 0.06 0.043 0.074 0.149 Hex2Cer(d28:0) 0.064 0.815 0.113 0.054 0.079 0.316 0.09 0.422 0.125 0.047 0.04 0.108 0.104 0.117 0.204 0.082 0.074 0.399 0.531 0.164 0.054 0.077 0.061 0.108 0.082 0.977 0.076 0.213 0.942 0.753 0.081 0.057 Hex2Cer(d36:1) 0.032 0.134 0.124 0.055 0.008 0.049 0.015 0.004 0.05 0.037 0.141 0.23 0.09 0.137 0.035 0.133 0 0.01 0.016 0.003 0 0 0.02 0.015 0.006 0.014 0.036 0.109 0.004 0.024 0 0.025 Hex2Cer(d42:2) 0.305 1.447 1.057 0.415 0.104 0.352 0.045 0.052 0.212 0.073 1.192 1.251 0.587 1.127 0.136 0.594 0.024 0.014 0.086 0.011 0.016 0.017 0.014 0.036 0.046 0.052 0.124 0.344 0.058 0.038 0.049 0.1 Hex2Cer(d43:0) 0.134 0.014 0.153 0.01 0.021 0.163 0.017 0.008 0.139 0.023 0.005 0.104 0.096 0.02 0.138 0.103 0.195 0.036 0.172 0.052 0.035 0.188 0.048 0.038 0.159 0.033 0.035 0.286 0.157 0.037 0.148 0.172 Hex2Cer(d44:2) 0.036 0.113 0.097 0.052 0.006 0.019 0 0 0.024 0 0.138 0.175 0.076 0.153 0.006 0.072 0 0 0 0 0 0 0 0 0 0 0 0.006 0 0.005 0 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name CE(16:0) CE(16:1) CE(18:1) CE(18:2) CE(20:4) CE(22:6) Cer(d24:0) Cer(d30:1) Cer(d31:3) Cer(d32:0) Cer(d34:0) Cer(d34:1) Cer(d36:0) Cer(d36:1) Cer(d36:2) Cer(d37:1) Cer(d38:1) Cer(d38:2) Cer(d40:1) Cer(d40:2) Cer(d41:1) Cer(d41:2) Cer(d42:1) Cer(d42:2) Cer(d42:3) Cer(d44:2) Hex1Cer(d33:1) Hex1Cer(d35:2) Hex1Cer(d36:1) Hex1Cer(d36:2) Hex1Cer(d38:1) Hex1Cer(d38:2) Hex1Cer(d40:1) Hex1Cer(d40:2) Hex1Cer(d41:1) Hex1Cer(d41:2) Hex1Cer(d42:1) Hex1Cer(d42:2) Hex1Cer(d42:3) Hex1Cer(d43:1) Hex1Cer(d44:1) Hex1Cer(d44:2) Hex1Cer(d44:3) Hex2Cer(d28:0) Hex2Cer(d36:1) Hex2Cer(d42:2) Hex2Cer(d43:0) Hex2Cer(d44:2) METABOLITES_END #END