Date of Award

Fall 2014

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

First Advisor

Weiguo Tao

Committee Member 1

Sandra Rossie

Committee Member 2

Mark Hall

Committee Member 3

Jin-Rong Xu

Abstract

Comprehensive protein-protein interaction network analysis can help reveal protein functions in a system-wide manner. A reliable knowledgebase of interaction networks is not only important for selecting the candidates for drug therapies, but also for evaluating the disease risk. In current interaction databases, 322579 interactions comprised of 56460 proteins have been reported (statistical analysis from APID: Agile Protein Interaction DataAnalyzer; http://bioinfow.dep.usal.es/apid/index.htm). The huge datasets are contributed mainly by yeast -two -hybrid (Y2H) screening and affinity-purification followed by mass spectrometry (AP-MS). High false positive rates and failing to cover certain interaction categories are the limitations of these two methods. Here, we developed two proteomic strategies to address the limitations. First, we attempt to expand the dynamic of protein abundance in peptides mixtures by identifying phosphorylated peptides. The phosphorylation is a ubiquitous post-translation modification which is involved in regulating the signaling pathway and protein-protein interaction. The step of phosphopeptide enrichment after immunoprecipitation was employed as the second purification to reduce the complexity of the samples. With this approach, the sample complexity was significantly reduced because most of the unmodified peptides were removed after the phosphopeptide enrichment. In this simplified sample, the number of many high abundant proteins and peptides identified, such as ribosomal proteins, was significantly reduced, resulting in improved identification of Plk1 interacting proteins. We also applied this approach to the PRMT5 network, in which few phosphorylation events have been reported. In the PRMT5 complex, we identified multiple phosphoproteins that were not identified in conventional AP-MS, which provided us with complementary candidates list for follow-up investigation. For the second strategy, we developed a trifunctional chemical reagent to study the virus-host interactome. The reagent contains a maleimide group for labeling virus surface proteins, a biotin tag for capturing the virus particle, and a TAMRA fluorescence molecule in the first generation reagent for method development. The maleimide group targets the free thiols on Sindbis virus surface proteins is the critical step to process the virus modification by the trifunctional reagent. In this study, we successfully captured the labeled-virus by streptavidin agarose beads from the infected cells, and several identified host proteins were known to involve in the virus infection pathways which means the labeled-viruses still maintain the pathogenic property. The method will further replace the TAMRA fluorescent beacon to a UV-activated photocrosslinker for "fixing" the virus binding partners after several harsh wash conditions to remove the non-specific binding protein. We expect the second generation reagent should result in more accurate identification of host proteins involved in virus infection.

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