Application of Affinity Chromatography Combined with Capillary Electrophoresis or Mass Spectrometry in the Biochemical Analysis

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Title: Application of Affinity Chromatography Combined with Capillary Electrophoresis or Mass Spectrometry in the Biochemical Analysis
Author: Zhou, Wei
Advisors: Morteza G. Khaledi, Chair
Charles B. Boss, Member
Edmond F. Bowden, Member
Bill Switzer, Member
Kenneth B. Tomer, Member
Abstract: The focus of the research has been to develop combined techniques, such as affinity chromatography combined with capillary electrophoresis or mass spectrometry, for the structural analysis of biologically important proteins. In the first part of the research, a method that allows the direct analysis the peptides affinity-bound to the immobilized metal ion media by matrix assisted laser desorption/ionization mass spectrometry (MALDI/MS) has been developed and applied to detect sequence errors of recombinant proteins occuring at the N-terminus and to locate phosphorylation sites in proteins. This method allows the fast identification of two recombinant proteins with expression errors, one is proteins p24, a major capsid protein of human immunodeficiency virus (HIV), the other is Vif, a viral infectivity factor required for the efficient transmission of free virus. Phsophorylation sites on proteins p53 and p21 that are involved in determining cellular response to DNA damage are also detected using this method. Huge gain in terms of selectivity, sensitivity and structural information are achieved with minimal sample consumption. In the second part of the research, affinity capillary electrophoresis (ACE) has been applied to evaluate biomolecular interactions, such as protein-drug and antibody-antigen interactions, and to better understand the interaction. ACE with laser induced fluorescence detection (LIF) is used to systematically evaluate binding between phosphorothioate oligodeoxynucleotides (Sd, potential anti-HIV drugs) and viral envelope glycoprotein HIV-1 gp120. The results show that the interaction has a strong dependence on the sulfur phosphorothioate backbone. Chain length and the sequence of Sd also affect the ability of binding to gp120. The results may provide useful information to clinical trial. ACE is also used to examine the effect of each residue of the epitope of HIV-1 capsid protein p24 on their affinity to an anti-p24 monoclonal antibody. Each amino acid within epitope is successively substituted by alanine, and the effect of the substitutes on their affinity for the antibody is examined by ACE. We are able to determine the relative importance of each amino acid within the epitope to the binding affinity of the peptide. The results provide a better understanding of these interactions. High separation power and ease of automation of ACE offer an effective and rapid means to study of these types of biological interaction.
Date: 2000-01-31
Degree: PhD
Discipline: Chemistry

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