Studies on the Extracellular Vesicle protein Cargo
Extracellular vesicles (EVs) are membrane-enclosed structures released by all cell types of the body, Because of their role in cell-cell communication they represent a new paradigm in biology. Exploration of their molecular structure is an exciting and pioneering basic science task. Their therapeutic and diagnostic use is still in its infancy, just beginning.
In the project we investigate the external and internal protein cargos of extracellular vesicles, and the information carried by these cargos in inflammatory and tumorous diseases.
Our earlier collaboration using mass spectrometry to analyze extracellular vesicles has revealed that besides canonical vesicular proteins numerous plasma proteins (e.g. albumin, transferrin, fibrinogen) and immune complex-associated proteins were present in the extracellular vesicle preparations. This suggests the above proteins represent the external protein cargo (protein corona) of extracellular vesicles. This raised the possibility that i) the vesicular protein cargo may substantially modify the effect of vesicles on cells and ii) may carry diagnostic information in inflammatory and tumorous diseases.
1. We isolate EV subpopulations from i) human cell lines, ii) healthy human blood plasma, iii) from blood plasma of rheumatoid arthritis patients and iv) from the plasma of patients with pancreas carcinoma. Quantitative and other characterization of extracellular vesicles will be carried out by using the methods applied routinely by our team (such as TRPS, tEM, WB, FACS, ELLBA). This will be followed by the nano-HPLC/MS(MS) analysis of extracellular vesicles. We optimize mass spectrometry for extracellular vesicles.
2. In order to investigate the external protein cargo of extracellular vesicles we use extensive washing of our vesicle preparations with i) physiological salt solution, ii) >1M salt solution iii) using limited trypsin digestion while monitoring the integrity of the vesicular membranes. We carry mass spectrometry after each procedure.
3. We isolate extracellular vesicle subpopulations from 24 h conditioned media of human cell lines in serum free medium. These ”nascent” vesicles are incubated in EV-depleted healthy, inflammatory and tumorous blood plasma followed by mass spectrometry.
4. We also investigate the effect of the external protein cargo on the effects extracellular vesicles on cells.
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