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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV110022	1/1	Homo sapiens	Urine	(d)(U)C DG	Staubach S	2011	33%
Study summary Full title Differential glycomics of epithelial membrane glycoproteins from urinary exovesicles reveals shifts toward complex-type N-glycosylation in classical galactosemia All authors Staubach S, Schadewaldt P, Wendel U, Nohroudi K, Hanisch FG Journal J Proteome Res Abstract A variety of genetic variations in the galactose-1-phosphate uridyltransferase (GALT) gene cause pro (show more...)A variety of genetic variations in the galactose-1-phosphate uridyltransferase (GALT) gene cause profound activity loss of the enzyme and acute toxic effects mediated by accumulating metabolic intermediates of galactose in newborns induced by dietary galactose. However, even on a severely galactose-restricted diet, patients develop serious long-term complications of the CNS and ovaries, which may result from damaging perturbations in cell biology caused by endogenously synthezised galactose. Under galactose stress, the cosubstrate of GALT, galactose-1-phosphate, accumulates and disturbs catabolic and anabolic pathways of the carbohydrate metabolism with potential effects on protein glycosylation and membrane localization of glycoprotein receptors, like the epidermal growth factor receptor. To address this issue in view of a cellular pathomechanism, we performed a differential semiquantitative N-glycomics study of membrane proteins. A suitable noninvasive cellular material derived from epithelial plasma membranes was found in urinary exovesicles and in the shed Tamm-Horsfall protein. By applying matrix-assisted laser ionization mass spectrometry on permethylated, PNGaseF released N-glycans, we demonstrate that GALT deficiency is associated with dramatic shifts from prevalent high-mannose-type glycans found in healthy subjects toward complex-type N-linked glycosylation in patients. These N-glycosylation shifts were observed on exosomal N-glycoproteins but not on the Tamm-Horsfall glycoprotein, which showed predominant high-mannose-type glycosylation with M6. (hide) EV-METRIC 33% (65th 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 Urine Sample origin NAY Focus vesicles Exovesicles 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 (d)(U)C DG Protein markers EV: Alix/ HSP70/ MUC1/ Flotillin2 non-EV: Tamm-Horsfall glycoprotein Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Wash: volume per pellet (ml) 1.5 Density gradient Only used for validation of main results Yes Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ HSP70/ Flotillin2/ MUC1 Detected contaminants Tamm-Horsfall glycoprotein ELISA Antibody details provided? No Detected EV-associated proteins Flotillin2/ MUC1 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

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EV-TRACK ID	EV110022
species	Homo sapiens
sample type	Urine
condition	NAY
separation protocol	(d)(U)C DG
Exp. nr.	1
EV-METRIC %	33