APPLICATIONS OF DYNAMIC ISOELECTRIC/ANISOTROPY BINDING LIGAND ASSAY FOR PROTEOMIC RESEARCH

Author

Hanna Elizabeth Pueblo, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2012

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Tolley, Luke

Abstract

The work presented in this dissertation centers around the development of analytical tools for the study of advanced proteomics. Section 1 of this work reviews the need for high efficiency protein separation techniques. Dynamic isoelectric focusing (DIEF) is new technique similar to capillary isoelectric focusing (CIEF) invented by Dr. Luke Tolley at Southern Illinois University Carbondale. Using DIEF, the electric field inside the separation capillary can be modified using high voltage electrodes, additional to the anode and cathode, to control the depth and shape of the resulting pH gradient. By changing the pH gradient, the location and width of focused protein bands can be controlled. As a new analytical technique, the development of DIEF required the design and fabrication of special holders which allow for electrical connections to be made at lengths along the separation capillary. These holders were also designed to have a removable section of capillary to extract very specific pH range proteins from high-resolution separations. Higher throughput DIEF systems were investigated, as well as multiplexed DIEF systems. Section 2 covers the topic of dynamic isoelectric/anisotropy ligand binding assay (DIABLA). DIABLA is a new method used to identify proteins in a complex sample that bind to a known molecule. DIABLA has the potential to be used in two complimentary ways, discovery mode and scanning mode. Both modes are accomplished by using DIEF, followed by fluorescence anisotropy as a sensitive detection method. This allows the entire length of capillary to be scanned to identify areas of non-zero anisotropy, which indicate binding interactions between the protein and target molecule. The binding protein(s) can then be extracted using the removable section of capillary from the DIEF holder, and can be identified by using a second dimension analysis, such as LC/MS/MS. DIABLA was verified in a series of proof-of-concept experiments in both discovery and scanning modes. These experiments involved fluorescently tagging proteins that were focused in the presence of a ligand tagged with a different fluorophore. The usefulness of DIABLA as a separation technique was demonstrated in four specific analyses of complex protein samples in Chapter 10.