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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV110096	1/1	Homo sapiens	Urine	(d)(U)C Filtration	Hiemstra TF	2011	29%
Study summary Full title Uromodulin exclusion list improves urinary exosomal protein identification All authors Hiemstra TF, Charles PD, Hester SS, Karet FE, Lilley KS Journal J Biomol Tech Abstract Advances in mass spectrometry (MS) have encouraged interest in its deployment in urine biomarker stu (show more...)Advances in mass spectrometry (MS) have encouraged interest in its deployment in urine biomarker studies, but success has been limited. Urine exosomes have been proposed as an ideal source of biomarkers for renal disease. However, the abundant urinary protein, uromodulin, cofractionates with exosomes during isolation and represents a practical contaminant that limits MS sensitivity. Uromodulin depletion has been attempted but is labor- and time-intensive and may remove important protein biomarkers. We describe the application of an exclusion list (ExL) of uromodulin-related peptide ions, coupled with high-sensitivity mass spectrometric analysis, to increase the depth of coverage of the urinary exosomal proteome. Urine exosomal protein samples from healthy volunteers were subjected to tandem MS and abundant uromodulin peptides identified. Samples were run for a second time, while excluding these uromodulin peptides from fragmentation to allow identification of peptides from lower-abundance proteins. Uromodulin exclusion was performed in addition to dynamic exclusion. Results from these two procedures revealed 222 distinct proteins from conventional analysis, compared with 254 proteins after uromodulin exclusion, of which 188 were common to both methods. By unmasking a previously unidentified protein set, adding the ExL increased overall protein identifications by 29.7% to a total of 288 proteins. A fixed ExL, used in combination with conventional methods, effectively increases the depth of urinary exosomal proteins identified by MS, reducing the need for uromodulin depletion. (hide) EV-METRIC 29% (60th 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 exosomes 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 Filtration Adj. k-factor 60.07 (pelleting) Protein markers EV: non-EV: Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 135 Pelleting: rotor type 45Ti Pelleting: adjusted k-factor 60.07 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis

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EV-TRACK ID	EV110096
species	Homo sapiens
sample type	Urine
condition	NAY
separation protocol	(d)(U)C Filtration
Exp. nr.	1
EV-METRIC %	29