Development of Techniques for Plasma Membrane and Cell Surface Enrichment

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Date
2014
Authors
Rose, Rebecca Leigh
Advisor
Fenselau, Catherine
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Abstract
The goal of this work is to develop and compare two methods of cell surface protein enrichment. The plasma membrane and cell surface proteomes are of great interest for the development of drug targets and increasing our understanding of communication pathways, especially in disease states. Here we evaluate the enrichment of the plasma membrane proteome utilizing the nanowire pellicle technique. The pellicle is constructed with iron silicate or silica core nanowires to observe the effects of wire density upon enrichment success. Comparison of these two wire pellicles reveals a two-fold enrichment of transmembrane proteins, over a multiple myeloma whole cell lysate, in both samples, with a slightly higher trend in the iron silicate nanowire pellicle. A covalent tagging method was also undertaken to assess its potential application to the study of myeloid-derived suppressor (MDSC) cell surfaces. This method couples glycan oxidation with biotinylation and enrichment on immobilized streptavidin to isolate surface glycoproteins. Application of this method to myeloid-derived suppressor cells yielded cell surface enrichment values ranging from approximately 50-60%. Furthermore, several protein groups of interest were identified for further assessment.
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