Graduation Semester and Year

2015

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Saiful M Chowdhury

Second Advisor

Purnendu K Dasgupta

Third Advisor

Peter Kroll

Abstract

Study of posttranslational modifications (PTMs) is very important for developing potential new drugs for the treatment of several diseases. Posttranslational lipid modifications are major PTMs which help proteins to anchor to cell membranes and facilitate protein-protein interactions. There are several kinds of lipid modifications of proteins. The major lipid modifications are palmitoylation, myristoylation and prenylation. Prenylation are of two types - farnesylation and geranylgeranylation. These modifications are involved with several human cancers and genetic diseases. Hutchinson-Gilford progeria syndrome (HGPS), a disease which causes premature aging for children is the product of prenylation. Understanding the mechanism of prenylation and their substrates is extremely needed to find the new drug target and develop new therapeutics for cancer and genetic diseases. Current identification methods for prenyl protein are laborious and time consuming and in some cases tailored to specific proteins. Most of the studies on protein prenylation use radioactive labeling, chemical reporters, LC-MS analysis and tagging via substrate method. None of the method can identify prenyl proteins globally, their sites and types in a single experiment. A global or large-scale method is needed to identify all the proteins which go through prenylation during cellular events. My dissertation work focused on the large scale identification of prenylated peptides/proteins by novel liquid-chromatography and mass spectrometric methods. We have developed mass spectrometry cleavable approaches to differentiate and locate the prenylation types. Prenyl proteins are hydrophobic and very difficult to analyze by LC-MS/MS. They have inconsistent gas phase fragmentation behavior during tandem mass spectrometry. We have studied in detail the CID and ETD mass spectrometry behavior of prenyl peptides. We have also converted prenyl peptides to more hydrophobic by oxidation chemistry. The fragmentation behavior of the synthesized prenylated peptides as well as the modified ones were studied in both collision induced dissociation (CID) and electron transfer dissociation (ETD) methods. On fragmentation, the loss of RSOH group (R = farnesyl/geranylgeranyl) from oxyprenylated peptides was found that result in the formation of a signature peak in the mass spectra. The mass loss distinguishes the type of prenylation. Oxidation chemistry was also incorporated epoxy groups in the prenyl side chain and increased the hydrophilicity and made them enrichable by chemical labeling method. In this dissertation, we developed a novel method to distinguish and locate a major lipid modification and we believe the study will significantly contribute in advancing cancer treatment and diagnosis.

Keywords

Mass spectrometry, Proteomics, Posttranslational modification, Prenylation, Farnesylation, Geranylgeranylation, Fragmentation

Disciplines

Chemistry | Physical Sciences and Mathematics

Comments

Degree granted by The University of Texas at Arlington

Included in

Chemistry Commons

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