From allergen detection to protein-ligand interactions

Fundamental biological and biochemical studies, proteomics, practical nutrition-related and medical applications, all the research fields, where bioanalytical chemistry is an essential tool, constantly require the improvement of existing techniques of bioanalysis together with the development of new ones. To successfully meet and solve emerging scientific challenges, modern bioanalytical methodologies should provide high sensitivity, selectivity and efficiency of the analysis in combination with low sample consumption, fast experimental procedure and high throughput. This thesis presents various methods and tools that have been developed to address different aspects of bioanalytical chemistry starting from allergen detection in hypoallergenic alimentary products for food safety control and finishing with protein-ligand interaction studies. In all techniques described below, mass spectrometry (MS) was employed as a detection method owing to its exceptional selectivity and sensitivity, paired with rapid analyte identification. Immunoaffinity capillary electrophoresis (IACE) using magnetic beads as immunosupport was coupled with matrix-assisted laser desorption/ionization (MALDI) MS to perform sensitive analysis of two major bovine milk allergens, beta-lactoglobulin and alpha-lactalbumin, for food safety and quality control. Then, the developed technique was extended to the performance of an effective component-resolved diagnostic of cow`s milk allergy using a tiny amount of patient blood serum, while offering the possibility of unusual allergen discovery and allergenomic studies. As sample pretreatment is the key step of all analytical procedures, a new on-chip technique named solid-phase extraction-gradient elution mass spectrometry (SPE-GEMS) was demonstrated for the effective sample purification, enrichment and fractionation followed by on-line electrospray ionization (ESI) MS detection. In this method, especially useful for proteomic studies, magnetic beads coated with reverse-phase chromatographic coating were used as SPE sorbent for the analysis of various peptide mixtures, providing an advantage of simple sorbent regeneration in a disposable manner. As a continuation of this project, described SPE-GEMS procedure was modified for processing more complex biological samples with their identification by tandem MS detection and, hence, was referred to as SPE-GEMS/MS. Due to the application of mesoporous silica shell magnetic core microspheres as a stationary phase, the new technique displayed high selectivity and sensitivity towards large hydrophobic peptides, which is a valuable characteristic for middle-down proteomic experiments. To ensure fast and high throughput experimental performance together with low sample consumption, the analytical procedure can be designed using droplet-based microfluidics. Therefore, a new way of coupling electrostatic-spray ionization (ESTASI) MS with a water-in-oil droplet system was realized via microchip with a spyhole. The developed system provided efficient analysis of the droplet content, without dilution or an additional oil removal step. It was successfully applied for the performance of single-phase reactions, as well as biphasic reactions, namely protein-ligand interaction between beta-lactoglobulin and acetate of liposoluble vitamin E.