Ion alternatively. While many bioanalytical approaches are accessible that reveal the basic functions of proteins inside a data set, none of these show complete signalling cascades. In this study, we aimed to choose and combine the relevant tools to reconstruct the mechanism of action of milk-EV. Solutions: Protein rotein interaction networks were produced from the widespread milk-EV proteome (367 proteins) using STRING. Functional enrichment evaluation in STRING was employed to establish which proteins were involved in any from the relevant biological processes studied within the in vitro assays. Then, the signalling pathways have been constructed employing Uniprot entries and their associated sources from the individual proteins, supplemented with a general literature search (like KEGG and MetaCore pathways). Finally, interacting proteins were linked to these pathways. Outcomes: Interestingly, person proteins and protein clusters may very well be linked to particular signalling events and their function (activation or inhibition) fitted the observed in vitro data. For instance, proteins have been identified that will stimulate the P38 migration pathway and cytoskeleton remodelling. Additionally, the milk-EV proteome contained a terrific quantity of proteins which are known to inhibit T cells by way of suppression of PI3K/ AKT, RAS/RAF and MAPK pathways. Depending on the specific pathway, regulation can take place early within the signalling cascade or throughout the entire signalling pathway. Summary/Conclusion: By integrating many bioanalytical approaches we have been in a position to determine relevant proteins and identify their action and position in distinct signalling pathways. As expected, milk-derived EV include a cluster of proteins of which their combined actions are most likely to regulate intercellular communication.PT03.New sample preparation process for exosome proteome analysis Zhigang Sui; Huiming Yuan; Lihua Zhang; Yukui Zhang Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (People’s Republic)PT03.Unravelling the mechanism of action of milk-derived EV by linking their proteome to relevant signalling pathways making use of an unbiased comprehensive bioinformatics approach Martijn J.C. van Herwijnen1; Marijke I. Zonneveld2; Soenita Goerdayal3; Esther N.M Nolte-‘t-Hoen4; Johan Garssen5; Maarten Altelaar3; Frank A. Redegeld5; Marca H.M. Wauben4 Nemo Like Kinase Proteins Formulation Utrecht University, Utrecht, The Netherlands; 2Autophagy lab, department of Radiotherapy, Grow – school for Oncology Developmental Biology, Maastricht University, Maastricht, The Netherlands; 3Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Analysis and Utrecht Institute for Pharmaceutical Sciences, Utrecht, The Netherlands; 4Department of Biochemistry Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands, Utrecht, The Netherlands; 5Division of Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands, Utrecht, The NetherlandsBackground: EV are multisignaling elements and their functionality is probably to take place in the combined actions of their constituents, instead of single molecules. Upon deciphering their functional effects in vitro, the significant challenge is usually to define which molecules are accountable for their mode of action. Previously, we’ve got published the human milk-derived EVBackground: Emerging evidences show that exosomes represent a rich supply of ILT-4 Proteins supplier biomarkers within the diagno.