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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100035	1/1	Homo sapiens	NAY	(d)(U)C DG IAF	Buschow SI	2010	44%
Study summary Full title MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis All authors Buschow SI, van Balkom BW, Aalberts M, Heck AJ, Wauben M, Stoorvogel W Journal Immunol Cell Biol Abstract Professional antigen-presenting cells secrete major histocompatibility complex class II (MHC II) car (show more...)Professional antigen-presenting cells secrete major histocompatibility complex class II (MHC II) carrying exosomes with unclear physiological function(s). Exosomes are first generated as the intraluminal vesicles (ILVs) of a specific type of multivesicular body, and are then secreted by fusion of this compartment with the plasma membrane. We have previously shown that in contrast to the sorting of MHC II at lysosomally targeted multivesicular bodies, sorting of MHC II into exosomes does not rely on MHC II ubiquitination. In search for proteins that drive the incorporation of MHC II into exosomes or functionally discriminate exosomal from plasma membrane MHC II, we first analyzed the total proteome of highly purified B cell-derived exosomes using sensitive and accurate mass spectrometry (MS), and identified 539 proteins, including known and not previously identified constituents. Using quantitative MS, we then identified a small subset of proteins that were specifically co-immunoprecipitated with MHC II from detergent-solubilized exosomes. These include HSC71, HSP90, 14-3-3?, CD20 and pyruvate kinase type M2 (PKM2), and we speculate on the functionality of their interaction with exosomal MHC II. (hide) EV-METRIC 44% (84th 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 DG IAF Adj. k-factor 362.8 (pelleting) Protein markers EV: CD81/ MHC2 non-EV: Proteomics yes EV density (g/ml) 1.150 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted 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) 60 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 362.8 Density gradient Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Immunoaffinity capture Selected surface protein(s) MHC2 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ MHC2 ELISA Antibody details provided? No Detected EV-associated proteins MHC2

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EV-TRACK ID	EV100035
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DG IAF
Exp. nr.	1
EV-METRIC %	44