Presentation Title

The Effect of Exercise on Protein Yield Available for Mass Spectrometry from Platelet Derived Microvesicles in Humans

Format of Presentation

Poster to be presented Friday March 31, 2017

Abstract

Microvesicles are small, membrane-bound vesicles with a size range of 100-1000 nm that are involved in intercellular communication and regulation of many critical biological processes. Many cell types release microvesicles including endothelial cells, platelets, and neurons. Microvesicles derived from endothelial cells and platelets are found circulating in blood and increases in circulating platelet-derived microvesicles have been observed in response to exercise. Platelets also release microvesicles when activated by factors such as thrombin, ADP, or collagen, but the mechanism behind platelet-derived microvesicles release and low level platelet activation during exercise is not well understood. This study attempts to replicate a previously described method of microvesicle isolation by Protein Organic Solvent Precipitation (PROSPR). Microvesicles were isolated from blood samples by centrifugation to remove cells from the plasma, followed by removal of soluble protein from cell-free plasma by precipitation with cold acetone, leaving microvesicles behind in suspension. Microvesicles were then dried by vacuum centrifugation and lysed in a urea buffer. A buffer exchange was performed on released proteins by overnight acetone precipitation. Whole proteins fractionation was performed by isoelectric focussing and fractions were transferred to an elution buffer for mass spectrometric analysis. Protein yield was determined after lysis, buffer exchange and fractionation using bicinchoninic acid protein assays.

Department

Biological Sciences

Faculty Advisor

Mark Rakabowchuk

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The Effect of Exercise on Protein Yield Available for Mass Spectrometry from Platelet Derived Microvesicles in Humans

Microvesicles are small, membrane-bound vesicles with a size range of 100-1000 nm that are involved in intercellular communication and regulation of many critical biological processes. Many cell types release microvesicles including endothelial cells, platelets, and neurons. Microvesicles derived from endothelial cells and platelets are found circulating in blood and increases in circulating platelet-derived microvesicles have been observed in response to exercise. Platelets also release microvesicles when activated by factors such as thrombin, ADP, or collagen, but the mechanism behind platelet-derived microvesicles release and low level platelet activation during exercise is not well understood. This study attempts to replicate a previously described method of microvesicle isolation by Protein Organic Solvent Precipitation (PROSPR). Microvesicles were isolated from blood samples by centrifugation to remove cells from the plasma, followed by removal of soluble protein from cell-free plasma by precipitation with cold acetone, leaving microvesicles behind in suspension. Microvesicles were then dried by vacuum centrifugation and lysed in a urea buffer. A buffer exchange was performed on released proteins by overnight acetone precipitation. Whole proteins fractionation was performed by isoelectric focussing and fractions were transferred to an elution buffer for mass spectrometric analysis. Protein yield was determined after lysis, buffer exchange and fractionation using bicinchoninic acid protein assays.