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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130038	1/1	Homo sapiens	NAY	(d)(U)C	Duijvesz D	2013	33%
Study summary Full title Proteomic profiling of exosomes leads to the identification of novel biomarkers for prostate cancer All authors Duijvesz D, Burnum-Johnson KE, Gritsenko MA, Hoogland AM, Vredenbregt-van den Berg MS, Willemsen R, Luider T, Paša-Tolic L, Jenster G Journal PLoS One Abstract BACKGROUND: Current markers for prostate cancer, such as PSA lack specificity. Therefore, novel biom (show more...)BACKGROUND: Current markers for prostate cancer, such as PSA lack specificity. Therefore, novel biomarkers are needed. Unfortunately, the complexity of body fluids often hampers biomarker discovery. An attractive alternative approach is the isolation of small vesicles, i.e. exosomes, ?100 nm, which contain proteins that are specific to the tissue from which they are derived and therefore can be considered as treasure chests for disease-specific biomarker discovery. MATERIALS AND METHODS: Exosomes were isolated from 2 immortalized primary prostate epithelial cells (PNT2C2 and RWPE-1) and 2 PCa cell lines (PC346C and VCaP) by ultracentrifugation. After tryptic digestion, proteomic analyses utilized a nanoLC coupled with an LTQ-Orbitrap operated in tandem MS (MS/MS) mode. Accurate Mass and Time (AMT) tag approach was employed for peptide identification and quantitation. Candidate biomarkers were validated by Western blotting and Immunohistochemistry. RESULTS: Proteomic characterization resulted in the identification of 248, 233, 169, and 216 proteins by at least 2 peptides in exosomes from PNT2C2, RWPE-1, PC346C, and VCaP, respectively. Statistical analyses revealed 52 proteins differently abundant between PCa and control cells, 9 of which were more abundant in PCa. Validation by Western blotting confirmed a higher abundance of FASN, XPO1 and PDCD6IP (ALIX) in PCa exosomes. CONCLUSIONS: Identification of exosomal proteins using high performance LC-FTMS resulted in the discovery of PDCD6IP, FASN, XPO1 and ENO1 as new candidate biomarkers for prostate cancer. (hide) EV-METRIC 33% (75th 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 Cell culture supernatant 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: Alix/ CD9 non-EV: Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 110 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ CD9 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV130038
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	33