Aptamers and G-quartet DNA in capillary electrochromatography and affinity matrix assisted laser desorption/ionization mass spectrometry.

摘要

Protein separation is an extremely important challenge in modern analytical chemistry and serves many applications in biochemistry, biotechnology, and proteomics. In the first section of this dissertation, a rapid, non-denaturing approach to protein separation using G-quartet DNA stationary phases in open-tubular capillary electrochromatography (OT-CEC) was explored for separating proteins that may be difficult to separate by conventional methods.; Initial investigations utilized a model system of albumins from different animal species. Spectroscopic methods suggest differential interactions between the albumins and the G-quartet phases. Results show that OT-CEC with the 2-Plane G-quartet stationary phase offers better resolution than capillary zone electrophoresis (CZE), and a control non-G-quartet DNA sequence. Further investigations with the G-Quartet stationary phase using the glycoprotein ovalbumin suggest that the DNA phase improves resolution of protein glycoforms in the sample under certain experimental conditions.; The second section of this dissertation describes the use of DNA aptamers as affinity agents for a simple, reusable, direct technique to capture and analyze target proteins using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Aptamers have significant advantages over antibodies including smaller size, reusability, and ease of development and production. This technique, termed aptamer-enhanced MALDI (ApE-MALDI), was shown to specifically bind a target protein from complex mixtures. Both the thrombin aptamer and the Immunoglobulin E aptamer were used; each allowing for reproducible, specific capture of the given target protein.; The third section of this dissertation describes two different projects that are related to the other work through the exploration of the G-quartet structure. The first project used a fluorescent dye to confirm the structure of the 4-Plane G-quartet on the surface of a capillary. The second project involves initial studies of G-quartet biogels for entrapment of living cells, with the goal of using the gels in the development of artificial organs and tissues.