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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200123	2/2	Homo sapiens	Serum	(d)(U)C Total exosome isolation	Jorge Gutierrez-Franco	2018	12%
Study summary Full title Characterization of B7H6, an endogenous ligand for the NK cell activating receptor NKp30, reveals the identity of two different soluble isoforms during normal human pregnancy All authors Jorge Gutierrez-Franco, Rodolfo Hernandez-Gutierrez, Miriam Ruth Bueno-Topete, Jesse Haramati, Rosa Elena Navarro-Hernandez, Marta Escarra-Senmarti, Natali Vega-Magaña, Alicia del Toro-Arreola, Ana Laura Pereira-Suarez, Susana del Toro-Arreola Journal Immunobiology Abstract B7H6, an endogenous ligand expressed on tumor cell surfaces, triggers NKp30-mediated activation of h (show more...)B7H6, an endogenous ligand expressed on tumor cell surfaces, triggers NKp30-mediated activation of human NK cells. In contrast, the release of soluble B7H6 has been proposed as a novel mechanism by which tumors might evade NK cell-mediated recognition. Since NK cells are critical for the maintenance of early pregnancy, it is not illogical that soluble B7H6 might also be an important factor in directing NK cell activity during normal pregnancy. Thus, this study was focused on the characterization of soluble B7H6 during the development of normal pregnancy. Serum samples were obtained from healthy pregnant women who were experiencing their second pregnancies (n =36). Additionally, 17 of these pregnant participants were longitudinally studied for the presence of B7H6 during their second and third trimesters. Age-matched healthy non-pregnant women served as controls (n =30). The presence of soluble B7H6 was revealed by Western blotting. A further characterization was performed using an immunoproteomic approach based on 2DE-Western blotting combined with MALDI-MS. The results show that sera from all pregnant women were characterized by the presence of two novel isoforms of B7H6, both with lower MW than the reported of 51 kDa. These isoforms were either a heavy (∼37 kDa) or a light isoform (∼30 kDa) and were mutually exclusive. N-glycosylation did not completely explain the different molecular weights exhibited by the two isoforms, as was demonstrated by enzymatic deglycosylation with PNGase F. The confirmation of the identity and molecular mass of each isoform indicates that B7H6, while maintaining the C- and N-termini, is most likely released during pregnancy by a mechanism distinct from proteolytic cleavage. We found that both isoforms, but mainly the heavier B7H6, were released via exosomes; and that the lighter isoform was also released in an exosome-free manner that was not observed in the heavy isoform samples. In conclusion, we find that soluble B7H6 is constitutively expressed during pregnancy and that, moreover, the soluble B7H6 is present in two new isoforms, which are released by exosomal and exosome-free mechanisms. (hide) EV-METRIC 12% (43rd 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 Healthy pregnant Focus vesicles Exosome 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 Commercial method Protein markers EV: CD63/ B7H6 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit Total exosome isolation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ B7H6

	EV200123	1/2	Homo sapiens	Serum	(d)(U)C Total exosome isolation	Jorge Gutierrez-Franco	2018	0%
Study summary Full title Characterization of B7H6, an endogenous ligand for the NK cell activating receptor NKp30, reveals the identity of two different soluble isoforms during normal human pregnancy All authors Jorge Gutierrez-Franco, Rodolfo Hernandez-Gutierrez, Miriam Ruth Bueno-Topete, Jesse Haramati, Rosa Elena Navarro-Hernandez, Marta Escarra-Senmarti, Natali Vega-Magaña, Alicia del Toro-Arreola, Ana Laura Pereira-Suarez, Susana del Toro-Arreola Journal Immunobiology Abstract B7H6, an endogenous ligand expressed on tumor cell surfaces, triggers NKp30-mediated activation of h (show more...)B7H6, an endogenous ligand expressed on tumor cell surfaces, triggers NKp30-mediated activation of human NK cells. In contrast, the release of soluble B7H6 has been proposed as a novel mechanism by which tumors might evade NK cell-mediated recognition. Since NK cells are critical for the maintenance of early pregnancy, it is not illogical that soluble B7H6 might also be an important factor in directing NK cell activity during normal pregnancy. Thus, this study was focused on the characterization of soluble B7H6 during the development of normal pregnancy. Serum samples were obtained from healthy pregnant women who were experiencing their second pregnancies (n =36). Additionally, 17 of these pregnant participants were longitudinally studied for the presence of B7H6 during their second and third trimesters. Age-matched healthy non-pregnant women served as controls (n =30). The presence of soluble B7H6 was revealed by Western blotting. A further characterization was performed using an immunoproteomic approach based on 2DE-Western blotting combined with MALDI-MS. The results show that sera from all pregnant women were characterized by the presence of two novel isoforms of B7H6, both with lower MW than the reported of 51 kDa. These isoforms were either a heavy (∼37 kDa) or a light isoform (∼30 kDa) and were mutually exclusive. N-glycosylation did not completely explain the different molecular weights exhibited by the two isoforms, as was demonstrated by enzymatic deglycosylation with PNGase F. The confirmation of the identity and molecular mass of each isoform indicates that B7H6, while maintaining the C- and N-termini, is most likely released during pregnancy by a mechanism distinct from proteolytic cleavage. We found that both isoforms, but mainly the heavier B7H6, were released via exosomes; and that the lighter isoform was also released in an exosome-free manner that was not observed in the heavy isoform samples. In conclusion, we find that soluble B7H6 is constitutively expressed during pregnancy and that, moreover, the soluble B7H6 is present in two new isoforms, which are released by exosomal and exosome-free mechanisms. (hide) EV-METRIC 0% (median: 13% 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 Exosome 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 Commercial method Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit Total exosome isolation Characterization: Protein analysis None Protein Concentration Method Not determined

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EV-TRACK ID	EV200123
species	Homo sapiens
sample type	Serum
condition	Healthy pregnant	Control condition
separation protocol	dUC Total exosome isolation	dUC Total exosome isolation
Exp. nr.	2	1
EV-METRIC %	12	0