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.

The work in the dissertation expands the applications of DIEF and describes the development of incorporating DIEF in a microfluidic chip to create a comprehensive proteomics tool. Proof-of-concept DIEF experiments have been done previously, so the focus of this work is to explore the capabilities of DIEF. Dynamic isoelectric focusing (DIEF) is a separation technique invented by Dr. Luke Tolley. It is similar to capillary isoelectric focusing except it uses four high voltage electrodes to form a pH gradient instead of only two. The additional two electrodes are able to manipulate the pH gradient resulting in selection of the region and of the range of pH within a pre-defined sampling or extraction point. One of the first applications described for DIEF was to isolate a single protein from a complex mixture. The protein isolated was a cellulase enzyme capable of degrading multiple cellulose materials over a wide range of environmental conditions. DIEF did isolate the protein in a pH span of 0.005 which is equivalent to 0.075% of the total pH range. Fractions were collected for sequencing analysis, but the fractions were contaminated with keratin both times. DIEF was also successfully performed in an open air channel. Though other electromigration techniques have been successfully done in open air channels, these techniques were severely time and pH limited. In contrast, DIEF in an open air channel is capable of using the entire 3-10 pH range and can perform isolations until the proteins are completely separated. The device developed was also an improvement on increasing sample capacity. The channel was significantly bigger than the traditional glass capillaries used. Since the channel was open, fraction collection was made simpler by collecting using a pipette. This work also demonstrated that DIEF can be made through the use of silicone molding compounds and polyurethane. The amount of milling needed is reduced, the pieces are produced quickly, and a single mold can produce several pieces. Machining pieces with fragile bits is not needed to be done as much since only one acrylic piece is required produce a mold. The mold can produce several polyurethane pieces. This fabrication method has proven useful for making DIEF holders. The next step was to make DIEF a truly comprehensive proteomic tool by incorporating it into a microfluidic chip. Multiple sample fractions are rapidly generated on chip through the use of multiple bubbles simultaneously injected into the separation channel. This stops the separation and, since each droplet is isolated from others by a bubble on each side, the protein peaks are not able to broaden. This novel use of digital microfluidics is still a work in progress, but the fundamentals have been demonstrated. The fabrication protocol for making molds and PDMS casts was developed using materials and procedures that can be done in a common laboratory environment. DIEF is a separation technique still in its infancy, with a wide variety of available applications. DIEF will continue to be tested in other areas and developed into a comprehensive proteomic tool.