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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV250044	1/2	Homo sapiens	Tissue	DG	Su H	2021	75%
Study summary Full title Characterization of brain-derived extracellular vesicle lipids in Alzheimer's disease. All authors Su H, Rustam YH, Masters CL, Makalic E, McLean CA, Hill AF, Barnham KJ, Reid GE, Vella LJ Journal J Extracell Vesicles Abstract Lipid dyshomeostasis is associated with the most common form of dementia, Alzheimer's disease (AD). (show more...)Lipid dyshomeostasis is associated with the most common form of dementia, Alzheimer's disease (AD). Substantial progress has been made in identifying positron emission tomography and cerebrospinal fluid biomarkers for AD, but they have limited use as front-line diagnostic tools. Extracellular vesicles (EVs) are released by all cells and contain a subset of their parental cell composition, including lipids. EVs are released from the brain into the periphery, providing a potential source of tissue and disease specific lipid biomarkers. However, the EV lipidome of the central nervous system is currently unknown and the potential of brain-derived EVs (BDEVs) to inform on lipid dyshomeostasis in AD remains unclear. The aim of this study was to reveal the lipid composition of BDEVs in human frontal cortex, and to determine whether BDEVs have an altered lipid profile in AD. Using semi-quantitative mass spectrometry, we describe the BDEV lipidome, covering four lipid categories, 17 lipid classes and 692 lipid molecules. BDEVs were enriched in glycerophosphoserine (PS) lipids, a characteristic of small EVs. Here we further report that BDEVs are enriched in ether-containing PS lipids, a finding that further establishes ether lipids as a feature of EVs. BDEVs in the AD frontal cortex offered improved detection of dysregulated lipids in AD over global lipid profiling of this brain region. AD BDEVs had significantly altered glycerophospholipid and sphingolipid levels, specifically increased plasmalogen glycerophosphoethanolamine and decreased polyunsaturated fatty acyl containing lipids, and altered amide-linked acyl chain content in sphingomyelin and ceramide lipids relative to CTL. The most prominent alteration was a two-fold decrease in lipid species containing anti-inflammatory/pro-resolving docosahexaenoic acid. The in-depth lipidome analysis provided in this study highlights the advantage of EVs over more complex tissues for improved detection of dysregulated lipids that may serve as potential biomarkers in the periphery. (hide) EV-METRIC 75% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Tissue Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Protein markers EV: TSG101/ syntenin/ Syntenin non-EV: BiP/ calnexin Proteomics no EV density (g/ml) 1.23 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Tissue Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.6M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 13.2 Sample volume (mL) 0.2 Orientation Top_-down Speed (g) 180000 Duration (min) 180 Fraction volume (mL) 1.2 Fraction processing Centrifugation Pelleting: volume per fraction 70 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) mg tissue per starting sample Western Blot Detected EV-associated proteins TSG101/ syntenin Not detected contaminants BiP/ calnexin Detected EV-associated proteins TSG101/ Syntenin Detected contaminants BiP/ calnexin Characterization: Lipid analysis Yes

	EV250044	2/2	Homo sapiens	Tissue	DG	Su H	2021	50%
Study summary Full title Characterization of brain-derived extracellular vesicle lipids in Alzheimer's disease. All authors Su H, Rustam YH, Masters CL, Makalic E, McLean CA, Hill AF, Barnham KJ, Reid GE, Vella LJ Journal J Extracell Vesicles Abstract Lipid dyshomeostasis is associated with the most common form of dementia, Alzheimer's disease (AD). (show more...)Lipid dyshomeostasis is associated with the most common form of dementia, Alzheimer's disease (AD). Substantial progress has been made in identifying positron emission tomography and cerebrospinal fluid biomarkers for AD, but they have limited use as front-line diagnostic tools. Extracellular vesicles (EVs) are released by all cells and contain a subset of their parental cell composition, including lipids. EVs are released from the brain into the periphery, providing a potential source of tissue and disease specific lipid biomarkers. However, the EV lipidome of the central nervous system is currently unknown and the potential of brain-derived EVs (BDEVs) to inform on lipid dyshomeostasis in AD remains unclear. The aim of this study was to reveal the lipid composition of BDEVs in human frontal cortex, and to determine whether BDEVs have an altered lipid profile in AD. Using semi-quantitative mass spectrometry, we describe the BDEV lipidome, covering four lipid categories, 17 lipid classes and 692 lipid molecules. BDEVs were enriched in glycerophosphoserine (PS) lipids, a characteristic of small EVs. Here we further report that BDEVs are enriched in ether-containing PS lipids, a finding that further establishes ether lipids as a feature of EVs. BDEVs in the AD frontal cortex offered improved detection of dysregulated lipids in AD over global lipid profiling of this brain region. AD BDEVs had significantly altered glycerophospholipid and sphingolipid levels, specifically increased plasmalogen glycerophosphoethanolamine and decreased polyunsaturated fatty acyl containing lipids, and altered amide-linked acyl chain content in sphingomyelin and ceramide lipids relative to CTL. The most prominent alteration was a two-fold decrease in lipid species containing anti-inflammatory/pro-resolving docosahexaenoic acid. The in-depth lipidome analysis provided in this study highlights the advantage of EVs over more complex tissues for improved detection of dysregulated lipids that may serve as potential biomarkers in the periphery. (hide) EV-METRIC 50% (10th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Tissue Sample origin Alzheimer's Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Density gradient Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.23 Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Tissue Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.6M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 13.2 Sample volume (mL) 0.2 Orientation Top_-down Speed (g) 180000 Duration (min) 180 Fraction volume (mL) 1.2 Fraction processing Centrifugation Pelleting: volume per fraction 70 Pelleting: speed (g) 100000 Characterization: Protein analysis None Protein Concentration Method BCA Protein Yield (µg) ug tissue Characterization: Lipid analysis Yes Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) ~100

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EV-TRACK ID	EV250044
species	Homo sapiens
sample type	Tissue
condition	Control condition	Alzheimer's
separation protocol	Density gradient	Density gradient
Exp. nr.	1	2
EV-METRIC %	75	50