Date of Award

Summer 2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

W. Andy Tao

Committee Chair

W. Andy Tao

Committee Member 1

Mary J. Wirth

Committee Member 2

Robert L Geahlen

Committee Member 3

Hilkka I. Kenttämaa

Abstract

In the quest of deciphering biological mechanisms, the understanding of proteins and their functions is a vital component. However the complexity of the proteome poses an immense challenge for analytical chemists to explore it comprehensively. Currently mass spectrometry is at the forefront of proteome analysis, providing great capabilities to couple with modern separation techniques for wide applications. One major objective of current proteomic technologies is to identify proteins of interest among high background, in particular from complex biological samples. A variety of techniques have been developed over the years to simplify protein samples, which are broadly classified as enrichment, high abundance protein depletion and sample fractionation. Metal ions play critical roles in most protein enzymes and their interactions with proteins have been well documented, but their applications in proteomics are underdeveloped. We have been developing and applying metal-protein interaction based approaches for proteomics studies due to their affordability, versatility and tunability. Previously, the Tao lab introduced Polymer-based Metal Affinity Capture with Ti(IV) (PolyMAC-Ti) for phosphopeptide enrichment in solution phase as opposed to commonly used solid phase extraction methods. In chapter 2 we discuss further extension of PolyMAC technology to PolyMAC-Zr and the use of complementary metal ion enrichment to study Syk dependent phosphorylation in B-cells. In chapter 3, we discuss a study to couple PolyMAC-Ti with anti-phosphotyrosine antibody to investigate Syk and Lyn dependent phosphorylation in B-cells. In chapter 4, we introduce a method to capture and fractionate proteins based on copper-protein interactions. Its unique application in enriching and fractionating secretomes was demonstrated by identifying low abundance cytokines and chemokines.

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