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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230598	1/3	Homo sapiens	Milk	(d)(U)C	Ten-Doménech, Isabel	2024	78%
Study summary Full title Normalization approaches for extracellular vesicle-derived lipidomic fingerprints – A human milk case study All authors Isabel Ten-Doménech, Victoria Ramos-Garcia, Abel Albiach-Delgado, Jose Luis Moreno-Casillas, Alba Moreno-Giménez, María Gormaz, Marta Gómez-Ferrer, Pilar Sepúlveda, Máximo Vento, Guillermo Quintás, Julia Kuligowski Journal Chemometrics and Intelligent Laboratory Systems Abstract Human milk (HM) extracellular vesicles (EVs) are nano-sized, cell-derived particles sheathed in a li (show more...)Human milk (HM) extracellular vesicles (EVs) are nano-sized, cell-derived particles sheathed in a lipid bilayer that encase specific cargo for delivery from mother to infant. The aim of this study was to expand our understanding of the lipidomic fingerprint of HM-EVs, with a specific focus on the impact of data normalization using simulated and experimental data obtained from the analysis of HM samples from mothers of preterm (N = 5) and term infants (N = 5), and a pool of donor human milk from 20 mothers (before and after pasteurization). EVs were isolated by multi-stage ultracentrifugation and characterized in terms of total protein content, total particle count and size, surface tetraspanin profile and protein markers, and morphology. Lipidomic analysis after single-phase extraction was performed by liquid chromatography mass spectrometry (LC-MS). The effect of widely used data normalization strategies (i.e., sample volume, particle count, protein content, total lipids signal) was compared. Results show that for the selection of the optimum normalization approach, the specific study aims, as well as the purity and homogeneity of size distribution of EV isolates should be considered. While normalization attending the particle number can be useful for between sample comparisons in EV populations with similar particle size, normalization to total lipid content is preferred when lipid contamination is encountered. Our findings exemplify the need for guidance with respect to data processing in LC-MS-based lipidomics studies of EVs. (hide) EV-METRIC 78% (86th 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 Milk Sample origin Mothers of preterm infants 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 (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD63/ CD81/ HSP70 non-EV: Calnexin Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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: rotor type Type 50.2 Ti Pelleting: speed (g) 108763 Wash: volume per pellet (ml) 25 Wash: time (min) 120 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 108763 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of EV isolate Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ HSP70 Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 168-238 EV concentration Yes Particle yield as number of particles per milliliter of EV isolate: 1.4E10-1.4E12 EM EM-type Transmission-EM Image type Close-up, Wide-field Other particle analysis name(1) ExoView Report type Mean Report size 58-70 EV-concentration Yes Particle yield as number of particles per milliliter of EV isolate: 6E07-3E08

	EV230598	2/3	Homo sapiens	Milk	(d)(U)C	Ten-Doménech, Isabel	2024	56%
Study summary Full title Normalization approaches for extracellular vesicle-derived lipidomic fingerprints – A human milk case study All authors Isabel Ten-Doménech, Victoria Ramos-Garcia, Abel Albiach-Delgado, Jose Luis Moreno-Casillas, Alba Moreno-Giménez, María Gormaz, Marta Gómez-Ferrer, Pilar Sepúlveda, Máximo Vento, Guillermo Quintás, Julia Kuligowski Journal Chemometrics and Intelligent Laboratory Systems Abstract Human milk (HM) extracellular vesicles (EVs) are nano-sized, cell-derived particles sheathed in a li (show more...)Human milk (HM) extracellular vesicles (EVs) are nano-sized, cell-derived particles sheathed in a lipid bilayer that encase specific cargo for delivery from mother to infant. The aim of this study was to expand our understanding of the lipidomic fingerprint of HM-EVs, with a specific focus on the impact of data normalization using simulated and experimental data obtained from the analysis of HM samples from mothers of preterm (N = 5) and term infants (N = 5), and a pool of donor human milk from 20 mothers (before and after pasteurization). EVs were isolated by multi-stage ultracentrifugation and characterized in terms of total protein content, total particle count and size, surface tetraspanin profile and protein markers, and morphology. Lipidomic analysis after single-phase extraction was performed by liquid chromatography mass spectrometry (LC-MS). The effect of widely used data normalization strategies (i.e., sample volume, particle count, protein content, total lipids signal) was compared. Results show that for the selection of the optimum normalization approach, the specific study aims, as well as the purity and homogeneity of size distribution of EV isolates should be considered. While normalization attending the particle number can be useful for between sample comparisons in EV populations with similar particle size, normalization to total lipid content is preferred when lipid contamination is encountered. Our findings exemplify the need for guidance with respect to data processing in LC-MS-based lipidomics studies of EVs. (hide) EV-METRIC 56% (76th 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 Milk Sample origin Mothers of term infants 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 (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD63/ CD81/ HSP70 non-EV: Calnexin Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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: rotor type Type 50.2 Ti Pelleting: speed (g) 108763 Wash: volume per pellet (ml) 25 Wash: time (min) 120 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 108763 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of EV isolate Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ HSP70 Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 168-238 EV concentration Yes Particle yield as number of particles per milliliter of EV isolate: 1.4E10-1.4E12 Other particle analysis name(1) ExoView Report type Mean Report size 58-70 EV-concentration Yes Particle yield as number of particles per milliliter of EV isolate: 6E07-3E08

	EV230598	3/3	Homo sapiens	Milk	(d)(U)C	Ten-Doménech, Isabel	2024	44%
Study summary Full title Normalization approaches for extracellular vesicle-derived lipidomic fingerprints – A human milk case study All authors Isabel Ten-Doménech, Victoria Ramos-Garcia, Abel Albiach-Delgado, Jose Luis Moreno-Casillas, Alba Moreno-Giménez, María Gormaz, Marta Gómez-Ferrer, Pilar Sepúlveda, Máximo Vento, Guillermo Quintás, Julia Kuligowski Journal Chemometrics and Intelligent Laboratory Systems Abstract Human milk (HM) extracellular vesicles (EVs) are nano-sized, cell-derived particles sheathed in a li (show more...)Human milk (HM) extracellular vesicles (EVs) are nano-sized, cell-derived particles sheathed in a lipid bilayer that encase specific cargo for delivery from mother to infant. The aim of this study was to expand our understanding of the lipidomic fingerprint of HM-EVs, with a specific focus on the impact of data normalization using simulated and experimental data obtained from the analysis of HM samples from mothers of preterm (N = 5) and term infants (N = 5), and a pool of donor human milk from 20 mothers (before and after pasteurization). EVs were isolated by multi-stage ultracentrifugation and characterized in terms of total protein content, total particle count and size, surface tetraspanin profile and protein markers, and morphology. Lipidomic analysis after single-phase extraction was performed by liquid chromatography mass spectrometry (LC-MS). The effect of widely used data normalization strategies (i.e., sample volume, particle count, protein content, total lipids signal) was compared. Results show that for the selection of the optimum normalization approach, the specific study aims, as well as the purity and homogeneity of size distribution of EV isolates should be considered. While normalization attending the particle number can be useful for between sample comparisons in EV populations with similar particle size, normalization to total lipid content is preferred when lipid contamination is encountered. Our findings exemplify the need for guidance with respect to data processing in LC-MS-based lipidomics studies of EVs. (hide) EV-METRIC 44% (67th 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 Milk Sample origin Pool of donors 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 (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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: rotor type Type 50.2 Ti Pelleting: speed (g) 108763 Wash: volume per pellet (ml) 25 Wash: time (min) 120 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 108763 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of EV isolate Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis Other particle analysis name(1) ExoView¨ Report type Mean Report size 58-70 EV-concentration Yes Particle yield as number of particles per milliliter of EV isolate: 6E07-3E08

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EV-TRACK ID	EV230598
species	Homo sapiens
sample type	Milk
condition	Mothers of preterm infants	Mothers of term infants	Pool of donors
separation protocol	dUC	dUC	dUC
Exp. nr.	1	2	3
EV-METRIC %	78	56	44