Capillary Electrophoresis - Mass Spectrometry for Bioanalysis

Abstract

Bioanalysis is a subdivision of analytical chemistry and deals with biological analytes such as metabolites, proteins, nucleic acids, small molecules, virus particles and entire cells. The rationale of my thesis was to achieve two goals: (i) develop a set of ready to use methods (ii) which are capable providing exact concentrations of analytes as well as kinetic and thermodynamic parameters of their interactions. To investigate interactions between biomolecules special conditions are required which do not interfere with the course if biomolecule interactions. Establishing these conditions and optimization of separation and detection parameters can be tedious and can take longer than actual analysis of samples. I developed a variety of Capillary Electrophoresis – Mass Spectrometry (CE-MS) methods suitable for bionalalysis. CE-MS establishes a new paradigm that separation methods together with MS detection can be used as comprehensive kinetic tools. Most previous attempts to use chromatography and electrophoresis for studying nucleic acid interactions were restricted to assuming slow or no equilibrium between reactants. Kinetic CE (KCE) shows that non-zero kinetics and structural dynamics must be taken into account when separation happens. KCE-MS could be a valuable supplement to IM-MS due to the separation of ions in solution according to their size-to-charge ratio. These methods allowed to reveal new facts about biomolecules and added novel data to the bank of the mankind knowledge. For the best of my knowledge, kinetic parameters for TG2 and thrombin G-quadruplex folding were reported for the first time. I developed a homogeneous method to determine kon, koff and Kd of fast and weak noncovalent interactions between multiple unlabeled ligands (small molecule drugs) and an oligosaccharide (α- or β-cyclodextrin) simultaneously in one capillary microreactor. It has been shown for the first time that KCE can be used to separate and detect the slowly interconverting open and closed conformations of human TG2. It allowed the first direct measurement of the Kd value for calcium binding. Sixteen new substrates were discovered for three aminotransferases (AAT, BCAT, and DAAT). In addition, Viral qCE showed a feasibility to analyse both the count of intact viral particles and sample nucleic acid contamination.