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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120152	1/1	Homo sapiens	Urine	(d)(U)C	Pisitkun T	2012	14%
Study summary Full title Application of systems biology principles to protein biomarker discovery: urinary exosomal proteome in renal transplantation All authors Pisitkun T, Gandolfo MT, Das S, Knepper MA, Bagnasco SM Journal Proteomics Clin Appl Abstract PURPOSE: In mass spectrometry (MS)-based studies to discover urinary protein biomarkers, an importan (show more...)PURPOSE: In mass spectrometry (MS)-based studies to discover urinary protein biomarkers, an important question is how to analyze the data to find the most promising potential biomarkers to be advanced to large-scale validation studies. Here, we describe a systems biology-based approach to address this question. EXPERIMENTAL DESIGN: We analyzed large-scale liquid chromatography-tandem mass spectrometry (LC-MS/MS) data of urinary exosomes from renal allograft recipients with biopsy-proven evidence of immunological rejection or tubular injury (TI). We asked whether bioinformatic analysis of urinary exosomal proteins can identify biological-process based protein groups that correlate with biopsy findings and whether the protein groups fit with general knowledge of the pathophysiological mechanisms involved. RESULTS: LC-MS/MS analysis of urinary exosomal proteomes identified more than 1000 proteins in each pathologic group. These protein lists were analyzed computationally to identify the Biological Process and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway terms that are significantly associated with each pathological group. Among the most informative terms for each group were: sodium ion transport for TI; immune response for all rejection; epithelial cell differentiation for cell-mediated rejection; and acute inflammatory response for antibody-mediated rejection. Based on these terms, candidate biomarkers were identified using a novel strategy to allow a dichotomous classification between different pathologic categories. CONCLUSIONS AND CLINICAL RELEVANCE: The terms and candidate biomarkers identified make rational connections to pathophysiological mechanisms, suggesting that the described bioinformatic approach will be useful in advancing large-scale biomarker identification studies toward a validation phase. (hide) EV-METRIC 14% (40th 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 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) 60 Characterization: Protein analysis

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EV-TRACK ID	EV120152
species	Homo sapiens
sample type	Urine
condition	NAY
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	14