Year of Publication

2014

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Arts and Sciences

Department

Chemistry

First Advisor

Dr. Bert C. Lynn

Abstract

Proteomics refers to global characterization of the full set of proteins present in a biological sample. Various analytical disciplines contribute to proteomics but mass spectrometry became method of choice for analysis of complex protein samples.

Mass spectrometry allows for high throughput analysis of the proteome but, moreover, it has the ability to acquire higher-order information such as post-translational modifications (PTM). Glycosylation is the most abundant PTM on eukaryotic proteins.

This dissertation will focus on method development for structural proteomics that will be utilized to explain the glycoproteome of obligate intracellular protozoan parasite Toxoplasma gondii as a model system.

Optimization of sample preparation is addressed in the first part of this dissertation. Sample preparation for mass spectrometry analysis is a critical step in the proteomics workflow because the quality and reproducibility of sample extraction and preparation significantly impacts the separation and identification capabilities of mass spectrometers. Also, there are problems unique to intracellular parasites as limited amount, host cell impurity and choice of the host. The additional obstacle is to extract only glycosylated proteins for which there is no one standard method. Here we report the optimal sample preparation method utilizing agarose bound Concanavalin A (Con A) beads to efficiently pull down glycoproteins, dialyze and analyze them using MuDPIT. This method was further enhanced by passing the non-retained protein fraction (first flow-through) through a second Con A column and then passing the second non-retained protein fraction (second flow-through) through the third Con A column (3 sequential pull-downs) yielding 394 benchmark proteins.

Glycoproteome of Toxoplasma gondii is not yet fully understood. However, evidence suggests that glycosylation could be essential for cyst formation and maintenance which is characteristic of chronic stage of disease. The focus of the second part of dissertation is to better understand the differences in glycoproteomes of tachizoites and tissue cysts. Cyst proteins pulled down using optimized sample preparation method that do not appear in the tachyzoites pulldowns could be critical elements in the structural stability of the tissue cyst.

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