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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220163	1/3	Homo sapiens	Serum	qEV70	Lauren A. Newman	2022	75%
Study summary Full title Addressing MISEV guidance using targeted LC-MS/MS: A method for the detection and quantification of extracellular vesicle-enriched and contaminant protein markers from blood All authors Lauren A. Newman, Zivile Useckaite, Andrew Rowland Journal Journal of Extracellular Biology Abstract Extracellular vesicles (EVs) are membrane-bound nanosized particles released by cells into bodily fl (show more...)Extracellular vesicles (EVs) are membrane-bound nanosized particles released by cells into bodily fluids containing an array of molecular cargo. Several characteristics, including stability and accessibility in biofluids such as blood and urine, make EVs and associated cargo attractive biomarkers and therapeutic tools. To promote robust characterisation of EV isolates, the minimal requirements for the study of extracellular vesicles (MISEV) guidelines recommend the analysis of proteins in EV samples, including positive EV-associated markers and negative contaminant markers based on commonly co-isolated components of the starting material. Western blot is conventionally used to address the guidelines; however, this approach is limited in terms of quantitation and throughput and requires larger volumes than typically available for patient samples. The increasing application of EVs as liquid biopsy in clinical contexts requires a high-throughput multiplexed approach for analysis of protein markers from small volumes of starting material. Here, we document the development and validation of a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantification of markers associated with EVs and non-vesicle contaminants from human blood samples. The assay was highly sensitive, requiring only a fraction of the sample consumed for immunoblots, fully quantitative and high throughput. Application of the assay to EVs isolated by size exclusion chromatography (SEC) and precipitation revealed differences in yield, purity and recovery of subpopulations. (hide) EV-METRIC 75% (98th 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 Serum 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 qEV70 Protein markers EV: CD81/ TSG101/ CD9 non-EV: Calnexin/ Albumin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit qEV70 Other Name other separation method qEV70 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD81/ TSG101 Not detected EV-associated proteins CD9 Detected contaminants Albumin Not detected contaminants Calnexin Other 2 Targeted LC-MS Detected EV-associated proteins CD9/ CD81/ TSG101 Detected contaminants Albumin/ Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 101.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.33E11 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 70

	EV220163	2/3	Homo sapiens	Serum	ExoQuick	Lauren A. Newman	2022	75%
Study summary Full title Addressing MISEV guidance using targeted LC-MS/MS: A method for the detection and quantification of extracellular vesicle-enriched and contaminant protein markers from blood All authors Lauren A. Newman, Zivile Useckaite, Andrew Rowland Journal Journal of Extracellular Biology Abstract Extracellular vesicles (EVs) are membrane-bound nanosized particles released by cells into bodily fl (show more...)Extracellular vesicles (EVs) are membrane-bound nanosized particles released by cells into bodily fluids containing an array of molecular cargo. Several characteristics, including stability and accessibility in biofluids such as blood and urine, make EVs and associated cargo attractive biomarkers and therapeutic tools. To promote robust characterisation of EV isolates, the minimal requirements for the study of extracellular vesicles (MISEV) guidelines recommend the analysis of proteins in EV samples, including positive EV-associated markers and negative contaminant markers based on commonly co-isolated components of the starting material. Western blot is conventionally used to address the guidelines; however, this approach is limited in terms of quantitation and throughput and requires larger volumes than typically available for patient samples. The increasing application of EVs as liquid biopsy in clinical contexts requires a high-throughput multiplexed approach for analysis of protein markers from small volumes of starting material. Here, we document the development and validation of a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantification of markers associated with EVs and non-vesicle contaminants from human blood samples. The assay was highly sensitive, requiring only a fraction of the sample consumed for immunoblots, fully quantitative and high throughput. Application of the assay to EVs isolated by size exclusion chromatography (SEC) and precipitation revealed differences in yield, purity and recovery of subpopulations. (hide) EV-METRIC 75% (98th 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 Serum 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 ExoQuick Protein markers EV: CD81/ TSG101/ CD9 non-EV: Calnexin/ Albumin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD81/ TSG101 Not detected EV-associated proteins CD9 Detected contaminants Albumin Not detected contaminants Calnexin Other 2 Targeted LC-MS Detected EV-associated proteins CD81/ TSG101 Not detected EV-associated proteins CD9 Detected contaminants Albumin Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 111.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.31E13 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 150

	EV220163	3/3	Homo sapiens	Serum	qEV35	Lauren A. Newman	2022	75%
Study summary Full title Addressing MISEV guidance using targeted LC-MS/MS: A method for the detection and quantification of extracellular vesicle-enriched and contaminant protein markers from blood All authors Lauren A. Newman, Zivile Useckaite, Andrew Rowland Journal Journal of Extracellular Biology Abstract Extracellular vesicles (EVs) are membrane-bound nanosized particles released by cells into bodily fl (show more...)Extracellular vesicles (EVs) are membrane-bound nanosized particles released by cells into bodily fluids containing an array of molecular cargo. Several characteristics, including stability and accessibility in biofluids such as blood and urine, make EVs and associated cargo attractive biomarkers and therapeutic tools. To promote robust characterisation of EV isolates, the minimal requirements for the study of extracellular vesicles (MISEV) guidelines recommend the analysis of proteins in EV samples, including positive EV-associated markers and negative contaminant markers based on commonly co-isolated components of the starting material. Western blot is conventionally used to address the guidelines; however, this approach is limited in terms of quantitation and throughput and requires larger volumes than typically available for patient samples. The increasing application of EVs as liquid biopsy in clinical contexts requires a high-throughput multiplexed approach for analysis of protein markers from small volumes of starting material. Here, we document the development and validation of a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantification of markers associated with EVs and non-vesicle contaminants from human blood samples. The assay was highly sensitive, requiring only a fraction of the sample consumed for immunoblots, fully quantitative and high throughput. Application of the assay to EVs isolated by size exclusion chromatography (SEC) and precipitation revealed differences in yield, purity and recovery of subpopulations. (hide) EV-METRIC 75% (98th 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 Serum 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 qEV35 Protein markers EV: CD81/ TSG101/ CD9 non-EV: Calnexin/ Albumin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit qEV35 Other Name other separation method qEV35 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD81/ TSG101 Not detected EV-associated proteins CD9 Detected contaminants Albumin Not detected contaminants Calnexin Other 2 Targeted LC-MS Detected EV-associated proteins CD9/ CD81/ TSG101 Detected contaminants Albumin/ Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 95.53 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.20E12 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 70

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EV-TRACK ID	EV220163
species	Homo sapiens
sample type	Serum
condition	Control condition
separation protocol	qEV70	ExoQuick	qEV35
Exp. nr.	1	2	3
EV-METRIC %	75	75	75