Isolation Strategies and Proteomic Characterization of Extracellular Vesicles

Abstract: “Extracellular vesicles” is the collective term used to describe vesicular entities that are released from cells into the extracellular environment. These vesicles are composed of a delineating lipid membrane and its cargo which can comprise of bioactive molecules such as lipids, RNA, DNA and proteins which can be shuttled between cells and thus function as a means of cell-to-cell communication. The aims of this thesis were to address how discrepancies in isolation procedure effects the isolate, to distinguish vesicular proteins from co-isolated proteins, to determine the proteome of tissue resident EVs in tumors of colorectal cancer patients and finally to develop a method for high quality vesicle isolates from blood plasma. We demonstrate that different rotor types will influence not only the yield of isolated vesicles, but also the purity. Furthermore, prolonged ultracentrifugation can up to a point produce higher yields at no apparent cost to purity. Even after purification of vesicles with a density gradient, however, there are proteins in the isolate whose vesicular nature can be questioned as they are susceptible to membrane-impermeable proteolytic digestion. Interestingly, proteolysis of perceived luminal motifs of transmembrane proteins suggests the existence of proteins with unconventional topological orientation within the membrane. We further illustrate that vesicles isolated directly from colorectal tumor tissue greatly differ from vesicles from corresponding healthy tissue in their proteomic makeup. Lastly, we demonstrate the possibility of attaining a highly purified vesicle isolate from blood plasma that is of high enough quality for relevant proteomic evaluation. In conclusion, we demonstrate how both yield and purity can be optimized in cultured samples as well as in complex biological samples.

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