DEVELOPMENT OF A PROTEOMIC STRATEGY FOR ANALYSIS OF PLASMA MEMBRANE PROTEINS

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dc.contributor.advisorFenselau, Catherineen_US
dc.contributor.authorChoksawangkarn, Waeowaleeen_US
dc.contributor.departmentBiochemistryen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2013-10-04T05:31:27Z
dc.date.available2013-10-04T05:31:27Z
dc.date.issued2013en_US
dc.description.abstractPlasma membrane (PM) proteins play crucial roles in cell signaling and communications, and they are the targets of more than two thirds of drugs currently under development. Studies on changes in protein content, quantity and modifications of the PM proteins indicate metabolic alteration of disease related cells; therefore, mass spectrometry-based proteomic studies may lead to improved understanding of the pathology, the characterization of novel biomarkers, and discovery of future drug targets. The main objectives of my research are to develop an effective enrichment strategy and to optimize the proteomic workflow for analysis of PM proteins from cells in suspension. Strategies were optimized with human multiple myeloma cells cultured in suspension, and optimized strategies were applied to study the PM proteome of myeloid-derived suppressor cells (MDSC) collected from an animal model. We focus on optimization of the cationic nanoparticle pellicle method for enrichment of PM proteins. The principle of this method is to attach cationic nanoparticles to the cell surface by electrostatic interaction between the positively charged nanoparticles and the negatively charged cell surface. Thus, the heavier coated-plasma membrane sheets can be separated more easily from cellular organelles by centrifugation after cell lysis. The isolated PM proteins were identified by LC-MS/MS analysis. We have also optimized the workflow for proteolysis to enhance identification of hydrophobic PM proteins. Our studies reveal that higher density nanoparticle pellicles provide higher enrichment efficiency of the PM proteins and that a procedure using digestion in the gel matrix enhances the analysis of highly hydrophobic proteins. The most effective enrichment technique and optimized proteomic procedures were applied to characterize the PM proteins from MDSC obtained from BALB/c mice carrying 4T1 mammary carcinomas. These cells are known to accumulate in individuals with cancer and suppress anti-tumor immunity. Their accumulation and activity are increased with heightened-levels of inflammation. Comparative studies of the PM proteins expressed in the cells derived from basal- and heightened- levels of inflammation were performed using the spectral counting method. This work reveals a set of protein candidates that have a high potential to be involved in the inflammation-driven immunosuppressive activity of the MDSC.en_US
dc.identifier.urihttp://hdl.handle.net/1903/14532
dc.subject.pqcontrolledBiochemistryen_US
dc.subject.pquncontrolledHPLC-mass spectrometryen_US
dc.subject.pquncontrolledmyeloid-derived suppressor cellsen_US
dc.subject.pquncontrollednanoparticle pelliclesen_US
dc.subject.pquncontrolledplasma membraneen_US
dc.subject.pquncontrolledproteomicsen_US
dc.subject.pquncontrolledspectral countingen_US
dc.titleDEVELOPMENT OF A PROTEOMIC STRATEGY FOR ANALYSIS OF PLASMA MEMBRANE PROTEINSen_US
dc.typeDissertationen_US

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