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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230583	1/3	Homo sapiens	Human neocortical neurons	Filtration SEC (non-commercial)	Anastasi F	2021	17%
Study summary Full title Proteomics Profiling of Neuron-Derived Small Extracellular Vesicles from Human Plasma: Enabling Single-Subject Analysis. All authors Anastasi F, Masciandaro SM, Carratore RD, Dell'Anno MT, Signore G, Falleni A, McDonnell LA, Bongioanni P Journal Int J Mol Sci Abstract Small extracellular vesicles have been intensively studied as a source of biomarkers in neurodegener (show more...)Small extracellular vesicles have been intensively studied as a source of biomarkers in neurodegenerative disorders. The possibility to isolate neuron-derived small extracellular vesicles (NDsEV) from blood represents a potential window into brain pathological processes. To date, the absence of sensitive NDsEV isolation and full proteome characterization methods has meant their protein content has been underexplored, particularly for individual patients. Here, we report a rapid method based on an immunoplate covalently coated with mouse monoclonal anti- antibody for the isolation and the proteome characterization of plasma-NDsEV from individual Parkinson's disease (PD) patients. We isolated round-shaped vesicles with morphological characteristics consistent with exosomes. On average, 349 ± 38 protein groups were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, 20 of which are annotated in the Human Protein Atlas as being highly expressed in the brain, and 213 were shared with a reference NDsEV dataset obtained from cultured human neurons. Moreover, this approach enabled the identification of 23 proteins belonging to the Parkinson disease KEGG pathway, as well as proteins previously reported as PD circulating biomarkers. (hide) EV-METRIC 17% (55th 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 Cell culture supernatant Sample origin Neuroepithelial stem cells 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 Filtration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Human neocortical neurons EV-harvesting Medium Serum free medium Separation Method Filtration steps 0.2 or 0.22 µm Size-exclusion chromatography Total column volume (mL) 24 Sample volume/column (mL) 0.2 Resin type Superose 6 Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database ProteomeXchange Consortium Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 70.5

	EV230583	2/3	Homo sapiens	Blood plasma	IAF	Anastasi F	2021	17%
Study summary Full title Proteomics Profiling of Neuron-Derived Small Extracellular Vesicles from Human Plasma: Enabling Single-Subject Analysis. All authors Anastasi F, Masciandaro SM, Carratore RD, Dell'Anno MT, Signore G, Falleni A, McDonnell LA, Bongioanni P Journal Int J Mol Sci Abstract Small extracellular vesicles have been intensively studied as a source of biomarkers in neurodegener (show more...)Small extracellular vesicles have been intensively studied as a source of biomarkers in neurodegenerative disorders. The possibility to isolate neuron-derived small extracellular vesicles (NDsEV) from blood represents a potential window into brain pathological processes. To date, the absence of sensitive NDsEV isolation and full proteome characterization methods has meant their protein content has been underexplored, particularly for individual patients. Here, we report a rapid method based on an immunoplate covalently coated with mouse monoclonal anti- antibody for the isolation and the proteome characterization of plasma-NDsEV from individual Parkinson's disease (PD) patients. We isolated round-shaped vesicles with morphological characteristics consistent with exosomes. On average, 349 ± 38 protein groups were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, 20 of which are annotated in the Human Protein Atlas as being highly expressed in the brain, and 213 were shared with a reference NDsEV dataset obtained from cultured human neurons. Moreover, this approach enabled the identification of 23 proteins belonging to the Parkinson disease KEGG pathway, as well as proteins previously reported as PD circulating biomarkers. (hide) EV-METRIC 17% (46th 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 Blood plasma 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 Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Size-exclusion chromatography Resin type Immunoaffinity capture Selected surface protein(s) L1CAM Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database ProteomeXchange Consortium Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 115 EM EM-type Transmission-EM Image type Wide-field

	EV230583	3/3	Homo sapiens	Blood plasma	IAF	Anastasi F	2021	17%
Study summary Full title Proteomics Profiling of Neuron-Derived Small Extracellular Vesicles from Human Plasma: Enabling Single-Subject Analysis. All authors Anastasi F, Masciandaro SM, Carratore RD, Dell'Anno MT, Signore G, Falleni A, McDonnell LA, Bongioanni P Journal Int J Mol Sci Abstract Small extracellular vesicles have been intensively studied as a source of biomarkers in neurodegener (show more...)Small extracellular vesicles have been intensively studied as a source of biomarkers in neurodegenerative disorders. The possibility to isolate neuron-derived small extracellular vesicles (NDsEV) from blood represents a potential window into brain pathological processes. To date, the absence of sensitive NDsEV isolation and full proteome characterization methods has meant their protein content has been underexplored, particularly for individual patients. Here, we report a rapid method based on an immunoplate covalently coated with mouse monoclonal anti- antibody for the isolation and the proteome characterization of plasma-NDsEV from individual Parkinson's disease (PD) patients. We isolated round-shaped vesicles with morphological characteristics consistent with exosomes. On average, 349 ± 38 protein groups were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, 20 of which are annotated in the Human Protein Atlas as being highly expressed in the brain, and 213 were shared with a reference NDsEV dataset obtained from cultured human neurons. Moreover, this approach enabled the identification of 23 proteins belonging to the Parkinson disease KEGG pathway, as well as proteins previously reported as PD circulating biomarkers. (hide) EV-METRIC 17% (46th 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 Blood plasma Sample origin Parkinson's disease patients 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 Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method Immunoaffinity capture Selected surface protein(s) L1CAM Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database ProteomeXchange Consortium Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 97 EM EM-type Transmission-EM Image type Wide-field

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EV-TRACK ID	EV230583
species	Homo sapiens
sample type	Cell culture	Blood plasma	Blood plasma
cell type	Human neocortical neurons	NA	NA
medium	Serum free medium	NA	NA
condition	Neuroepithelial stem cells	Control condition	Parkinson's disease patients
separation protocol	Filtration/ Size-exclusion chromatography (non-commercial)	IAF capture (non-commercial)	IAF capture (non-commercial)
Exp. nr.	1	2	3
EV-METRIC %	17	17	17