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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190069	1/4	Homo sapiens	PC3	DG (d)(U)C	Mariscal, Javier	2020	67%
Study summary Full title Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles All authors Javier Mariscal, Tatyana Vagner, Minhyung Kim, Bo Zhou, Andrew Chin, Mandana Zandian, Michael R Freeman, Sungyong You, Andries Zijlstra, Wei Yang, Dolores Di Vizio Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer p (show more...)Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers. (hide) EV-METRIC 67% (94th 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 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 DG (d)(U)C Protein markers EV: CD81/ TSG101/ CD9/ HSPA5/ KRT18 non-EV: Proteomics no EV density (g/ml) 1.1 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PC3 EV-harvesting Medium Serum free medium Cell viability (%) 99 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type SW 28 Pelleting: speed (g) 10000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 30% Total gradient volume, incl. sample (mL) 16.2 Sample volume (mL) 0.2 Orientation Bottom-up Rotor type SW 28 Speed (g) 100000 Duration (min) 230 Fraction volume (mL) 2.5 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Other;Pierce 660nm Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins HSPA5/ KRT18 Not detected EV-associated proteins CD81/ TSG101/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 1770 EV concentration Yes

	EV190069	2/4	Homo sapiens	PC3	DG (d)(U)C	Mariscal, Javier	2020	67%
Study summary Full title Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles All authors Javier Mariscal, Tatyana Vagner, Minhyung Kim, Bo Zhou, Andrew Chin, Mandana Zandian, Michael R Freeman, Sungyong You, Andries Zijlstra, Wei Yang, Dolores Di Vizio Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer p (show more...)Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers. (hide) EV-METRIC 67% (94th 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 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 DG (d)(U)C Protein markers EV: TSG101/ CD81/ CD9/ HSPA5/ KRT18 non-EV: Proteomics no EV density (g/ml) 1.1 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PC3 EV-harvesting Medium Serum free medium Cell viability (%) 99 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 30% Total gradient volume, incl. sample (mL) 16.2 Sample volume (mL) 0.2 Orientation Bottom-up Rotor type SW 28 Speed (g) 100000 Duration (min) 230 Fraction volume (mL) 2.5 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Other;Pierce 660nm Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ TSG101/ CD81 Not detected EV-associated proteins HSPA5/ KRT18 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 131 EV concentration Yes

	EV190069	3/4	Homo sapiens	DU145	DG (d)(U)C	Mariscal, Javier	2020	56%
Study summary Full title Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles All authors Javier Mariscal, Tatyana Vagner, Minhyung Kim, Bo Zhou, Andrew Chin, Mandana Zandian, Michael R Freeman, Sungyong You, Andries Zijlstra, Wei Yang, Dolores Di Vizio Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer p (show more...)Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers. (hide) EV-METRIC 56% (90th 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 DIAPH3 Knock down 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 DG (d)(U)C Protein markers EV: CD9/ HSPA5 non-EV: Proteomics no EV density (g/ml) 1.1 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DU145 EV-harvesting Medium Serum free medium Cell viability (%) 99 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type SW 28 Pelleting: speed (g) 10000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 30% Total gradient volume, incl. sample (mL) 16.2 Sample volume (mL) 0.2 Orientation Bottom-up Rotor type SW 28 Speed (g) 100000 Duration (min) 230 Fraction volume (mL) 2.5 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method Other;Pierce 660 nm Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins HSPA5 Not detected EV-associated proteins CD9 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 1650 EV concentration Yes

	EV190069	4/4	Homo sapiens	DU145	DG (d)(U)C	Mariscal, Javier	2020	56%
Study summary Full title Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles All authors Javier Mariscal, Tatyana Vagner, Minhyung Kim, Bo Zhou, Andrew Chin, Mandana Zandian, Michael R Freeman, Sungyong You, Andries Zijlstra, Wei Yang, Dolores Di Vizio Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer p (show more...)Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers. (hide) EV-METRIC 56% (90th 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 DIAPH3 Knock down 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 DG (d)(U)C Protein markers EV: CD9/ HSPA5 non-EV: Proteomics no EV density (g/ml) 1.1 Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DU145 EV-harvesting Medium Serum free medium Cell viability (%) 99 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 30% Total gradient volume, incl. sample (mL) 16.2 Sample volume (mL) 0.2 Orientation Bottom-up Rotor type SW 28 Speed (g) 100000 Duration (min) 230 Fraction volume (mL) 2.5 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Other;Pierce 660 nm Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9 Not detected EV-associated proteins HSPA5 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Modus Reported size (nm) 116 EV concentration Yes

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EV-TRACK ID	EV190069
species	Homo sapiens
sample type	Cell culture
cell type	PC3	PC3	DU145	DU145
condition	Control condition	Control condition	DIAPH3 Knock down	DIAPH3 Knock down
separation protocol	DG (d)(U)C	DG (d)(U)C	DG (d)(U)C	DG (d)(U)C
Exp. nr.	1	2	3	4
EV-METRIC %	67	67	56	56