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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV190018	3/8	Homo sapiens	Cell culture supernatant	(Differential) (ultra)centrifugation Filtration Density gradient	Freitas D	2019	100%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 100% (99th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (Differential) (ultra)centrifugation + Filtration + Density gradient Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) 1.05-1.10 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.8 Sample volume (mL) 5.5 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Ultrafiltration Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins Alix/ CD9/ CD63/ HSP70/ Syntenin-1 Not detected EV-associated proteins CD81 Detected contaminants Albumin Proteomics database No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 131.65 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190018	7/8	Homo sapiens	Cell culture supernatant	(Differential) (ultra)centrifugation Filtration Density gradient	Freitas D	2019	100%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 100% (99th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 COSMC KO Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (Differential) (ultra)centrifugation + Filtration + Density gradient Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) 1.05-1.10 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition COSMC KO EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Density gradient Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.8 Sample volume (mL) 5.5 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Ultrafiltration Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins Alix/ Syntenin-1/ CD9/ CD63/ HSP70/ CD81 Detected contaminants Albumin Proteomics database No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 121.05 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190018	1/8	Homo sapiens	Cell culture supernatant	(Differential) (ultra)centrifugation Filtration	Freitas D	2019	75%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 75% (96th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (Differential) (ultra)centrifugation + Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 960 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 38 Wash: time (min) 120 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins CD9/ CD63/ Syntenin-1/ HSP70/ Alix/ CD81 Not detected EV-associated proteins Proteins regarded in the experiment as potentially EV-associated and not detected in the EVs Detected contaminants Albumin Not detected contaminants Cytochrome C Proteomics database No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 140.97 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190018	2/8	Homo sapiens	Cell culture supernatant	(Differential) (ultra)centrifugation Filtration Commercial method	Freitas D	2019	75%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 75% (96th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (Differential) (ultra)centrifugation + Filtration + Commercial method Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Filtration steps 0.22µm or 0.2µm Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins CD63/ Syntenin-1/ HSP70/ CD81 Not detected EV-associated proteins CD9/ Alix Detected contaminants Albumin Not detected contaminants Cytochrome C Proteomics database No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 148.3 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190018	4/8	Homo sapiens	Cell culture supernatant	(Differential) (ultra)centrifugation Filtration Commercial method	Freitas D	2019	75%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 75% (96th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Control condition Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (Differential) (ultra)centrifugation + Filtration + Commercial method Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Filtration steps 0.22µm or 0.2µm Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins Syntenin-1/ CD9/ CD63/ HSP70/ CD81 Not detected EV-associated proteins Alix Detected contaminants Albumin Proteomics database No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 138.37 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190018	5/8	Homo sapiens	Cell culture supernatant	(Differential) (ultra)centrifugation Filtration	Freitas D	2019	75%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 75% (96th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 COSMC KO Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (Differential) (ultra)centrifugation + Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition COSMC KO EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 960 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 38 Wash: time (min) 120 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins Syntenin-1/ CD9/ CD63/ HSP70/ Alix/ CD81 Detected contaminants Albumin Proteomics database No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 141.43 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190018	6/8	Homo sapiens	Cell culture supernatant	(Differential) (ultra)centrifugation Filtration Commercial method	Freitas D	2019	75%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 75% (96th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 COSMC KO Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (Differential) (ultra)centrifugation + Filtration + Commercial method Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition COSMC KO EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Filtration steps 0.22µm or 0.2µm Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins Alix/ Syntenin-1/ CD63/ HSP70/ CD81 Not detected EV-associated proteins CD9 Detected contaminants Albumin Proteomics database No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 152.5 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190018	8/8	Homo sapiens	Cell culture supernatant	(Differential) (ultra)centrifugation Filtration Commercial method	Freitas D	2019	75%
Study summary Full title Different isolation approaches lead to diverse glycosylated extracellular vesicle populations. All authors Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA. Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide) EV-METRIC 75% (96th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 COSMC KO Focus vesicles extracellular vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (Differential) (ultra)centrifugation + Filtration + Commercial method Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition COSMC KO EV-producing cells MKN45 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Filtration steps 0.22µm or 0.2µm Commercial kit qEV Characterization: Protein analysis Protein Concentration Method Other;silver staining Western Blot Detected EV-associated proteins Alix/ Syntenin-1/ CD9/ CD63/ HSP70/ CD81 Detected contaminants Albumin Proteomics database No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 148.33 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV190018
species	Homo sapiens
sample type	Cell culture
cell type	MKN45
condition	Control condition	COSMC KO	Control condition	Control condition	Control condition	COSMC KO	COSMC KO	COSMC KO
isolation protocol	dUC Filtration Density gradient	dUC Filtration Density gradient	dUC Filtration	dUC Filtration Commercial method	dUC Filtration Commercial method	dUC Filtration	dUC Filtration Commercial method	dUC Filtration Commercial method
Exp. nr.	3	7	1	2	4	5	6	8
EV-METRIC %	100	100	75	75	75	75	75	75

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