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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130240	1/1	Homo sapiens	NAY	(d)(U)C Filtration UF	Villarreal L	2013	14%
Study summary Full title Unconventional secretion is a major contributor of cancer cell line secretomes All authors Villarreal L, Méndez O, Salvans C, Gregori J, Baselga J, Villanueva J Journal Mol Cell Proteomics Abstract A challenge in achieving optimal management of cancer is the discovery of secreted biomarkers that r (show more...)A challenge in achieving optimal management of cancer is the discovery of secreted biomarkers that represent useful surrogates for the disease and could be measured noninvasively. Because of the problems encountered in the proteomic interrogation of plasma, secretomes have been proposed as an alternative source of tumor markers that might be enriched with secreted proteins relevant to the disease. However, secretome analysis faces analytical challenges that interfere with the search for true secreted tumor biomarkers. Here, we have addressed two of the main challenges of secretome analysis in comparative discovery proteomics. First, we carried out a kinetics experiment whereby secretomes and lysates of tumor cells were analyzed to monitor cellular viability during secretome production. Interestingly, the proteomic signal of a group of secreted proteins correlated well with the apoptosis induced by serum starvation and could be used as an internal cell viability marker. We then addressed a second challenge relating to contamination of serum proteins in secretomes caused by the required use of serum for tumor cell culture. The comparative proteomic analysis between cell lines labeled with SILAC showed a number of false positives coming from serum and that several proteins are both in serum and being secreted from tumor cells. A thorough study of secretome methodology revealed that under optimized experimental conditions there is a substantial fraction of proteins secreted through unconventional secretion in secretomes. Finally, we showed that some of the nuclear proteins detected in secretomes change their cellular localization in breast tumors, explaining their presence in secretomes and suggesting that tumor cells use unconventional secretion during tumorigenesis. The unconventional secretion of proteins into the extracellular space exposes a new layer of genome post-translational regulation and reveals an untapped source of potential tumor biomarkers and drug targets. (hide) EV-METRIC 14% (44th 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 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 UF Protein markers EV: 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis

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EV-TRACK ID	EV130240
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C Filtration UF
Exp. nr.	1
EV-METRIC %	14