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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210242	1/2	Homo sapiens	Blood plasma	(d)(U)C SEC (non-commercial) Filtration	Jayabalan N	2019	45%
Study summary Full title Quantitative Proteomics by SWATH-MS Suggest an Association Between Circulating Exosomes and Maternal Metabolic Changes in Gestational Diabetes Mellitus. All authors Jayabalan N, Lai A, Nair S, Guanzon D, Scholz-Romero K, Palma C, McIntyre HD, Lappas M, Salomon C Journal Proteomics Abstract Several factors including placental hormones (PH) released from the human placenta have been associa (show more...)Several factors including placental hormones (PH) released from the human placenta have been associated with the development of insulin resistance and gestational diabetes mellitus (GDM). However, circulating levels of PH does not correlate well with maternal insulin sensitivity across gestation, suggesting that other, previously unrecognized, mechanisms may be involved. The levels of circulating exosomes are higher in GDM compared to normal. GDM derived exosomes produce greater release of pro-inflammatory cytokines from endothelial cells compared to exosomes from normal, suggesting that their contents may differ compared to normal pregnancies. Using a quantitative, information-independent acquisition (Sequential Windowed Acquisition of All Theoretical Mass Spectra [SWATH]) approach, differentially abundant circulating exosome proteins are identified in women with normal glucose tolerance (NGT) and GDM at the time of GDM diagnosis. A total of 78 statistically significant proteins in the relative expression of exosomal proteins in GDM are compared with NGT. Bioinformatic analysis shows that the exosomal proteins in GDM target pathways are mainly associated with energy production, inflammation, and metabolism. Finally, an independent cohort of patients is used to validate some of the proteins identified by SWATH. The data obtained may be of utility in elucidating the underlying physiological mechanisms associated with insulin resistance in GDM. (hide) EV-METRIC 45% (78th 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 Blood plasma 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 Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: CD9/ TSG101/ CD63 non-EV: Grp94 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma 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: time(min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ TSG101/ CD63 Detected contaminants Grp94 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 105 +/- 15nm EV concentration Yes Particle yield No: 0.00e+0

	EV210242	2/2	Homo sapiens	Blood plasma	(d)(U)C SEC (non-commercial) Filtration	Jayabalan N	2019	45%
Study summary Full title Quantitative Proteomics by SWATH-MS Suggest an Association Between Circulating Exosomes and Maternal Metabolic Changes in Gestational Diabetes Mellitus. All authors Jayabalan N, Lai A, Nair S, Guanzon D, Scholz-Romero K, Palma C, McIntyre HD, Lappas M, Salomon C Journal Proteomics Abstract Several factors including placental hormones (PH) released from the human placenta have been associa (show more...)Several factors including placental hormones (PH) released from the human placenta have been associated with the development of insulin resistance and gestational diabetes mellitus (GDM). However, circulating levels of PH does not correlate well with maternal insulin sensitivity across gestation, suggesting that other, previously unrecognized, mechanisms may be involved. The levels of circulating exosomes are higher in GDM compared to normal. GDM derived exosomes produce greater release of pro-inflammatory cytokines from endothelial cells compared to exosomes from normal, suggesting that their contents may differ compared to normal pregnancies. Using a quantitative, information-independent acquisition (Sequential Windowed Acquisition of All Theoretical Mass Spectra [SWATH]) approach, differentially abundant circulating exosome proteins are identified in women with normal glucose tolerance (NGT) and GDM at the time of GDM diagnosis. A total of 78 statistically significant proteins in the relative expression of exosomal proteins in GDM are compared with NGT. Bioinformatic analysis shows that the exosomal proteins in GDM target pathways are mainly associated with energy production, inflammation, and metabolism. Finally, an independent cohort of patients is used to validate some of the proteins identified by SWATH. The data obtained may be of utility in elucidating the underlying physiological mechanisms associated with insulin resistance in GDM. (hide) EV-METRIC 45% (78th 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 Blood plasma Sample origin Pregnant with gestational diabetes mellitus 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 Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: CD9/ TSG101/ CD63 non-EV: Grp94 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma 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: time(min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ TSG101/ CD63 Detected contaminants Grp94 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 102 +/- 17nm EV concentration Yes Particle yield No: 0.00e+0

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EV-TRACK ID	EV210242
species	Homo sapiens
sample type	Blood plasma
condition	Healthy pregnant	Pregnant with gestational diabetes mellitus
separation protocol	dUC/ Size-exclusion chromatography (non-commercial)/ Filtration	dUC/ Size-exclusion chromatography (non-commercial)/ Filtration
Exp. nr.	1	2
EV-METRIC %	45	45