Projects
Comprehensive top-down-bottom-up proteomics
Czech Science Foundation, standard project 23-07581S (2023 – 2025).
There are two directions allowing characterization of the proteins: (i) top-down approach where the proteins themselves are separated and detected, or (ii) bottom-up approach focusing on the analysis of tryptic peptides and subsequent reconstruction of protein structure and function.
By integrating enzymatic digestion in the modulation interface, this project aims the development of comprehensive two-dimensional chromatographic system combining top-down separation of native proteins followed by the bottom-up analysis of their tryptic peptides.
Hypecrosslinked monolithic capillary and open tubular columns will be prepared with tailored pore size distribution allowing both size-based separation of the proteins and high-throughput and highefficiency separations of peptides.
Obtained results will provide beneficial information allowing a deeper understanding of the role of individual proteins in various biological systems.
Segmented two-dimensional liquid chromatography
Czech Science Foundation, standard project 20-21903S (2020 – 2022).
Multidimensional separations with more than one retention mechanism involved are being routinely applied in an analysis of complex samples. The most common hyphenation in liquid phases is two-dimensional liquid chromatography (2D LC). Here, all eluate of the first column is analyzed in the second column providing different retention mechanisms. However, special attention to sample transfer, its dilution, and speed of the second dimension must be considered.
The aim of the presented project is a development of 2D LC utilizing inline coupling of column segments in the first dimension. Developed spatial separation is then transferred to the second dimension, where another retention mechanism separates compounds eluted from the first dimension.
The experimental setup might be viewed as an online combination of a thin layer chromatography with a conventional column liquid chromatography.
An application of presented protocol in the two-dimensional separation of small and large molecules is outlined.
The project is an initial step in the further development of analytical systems for proteomics analysis.
Tailoring selectivity of polymer-based monolithic stationary phases
Czech Science Foundation, standard project 17-11252S (2017 – 2019).
In this project, preparation and characterization of the monolithic capillary column with enhanced selectivity for dopamine and its metabolites is proposed. UV-initiated grafting will be used to control the crosslink density of the polymer surface.
Further, development of multiretentive monolithic capillary column is proposed. In this case, the monolithic surface will be subsequently modified with several functional groups that will control the retention of small polar molecules. Chromatographic characterization and designs of experiments will be utilized to describe retention mechanisms on multiretentive monolithic capillary column.
Prepared columns will be used in the chromatographic screening of dopamine metabolism-related diseases, such as depression, schizophrenia, or Parkinson disease.
Development of a multifunctional monolithic capillary column with integrated sample focusing, separation, and on-column electrochemical detection
Czech Science Foundation, standard project 14-22426S (2014 – 2016).
In this project, we propose preparation of a multifunctional capillary device based on organic polymer monoliths. We propose spatially constrained surface modification of prepared monolithic columns to integrate sample focusing, separation, and on-column electrochemical detection. The surface grafting will be used to prepare segments with different polarity and chemistry. Monolithic surface will be also modified with polymers responding to external stimuli (e.g. temperature) to prepare so-called smart stationary phases. An electrochemical detector will be directly incorporated into the porous structure of monolithic column, either by embedding the microelectrodes at the end of the capillary column or by preparation of desired electrodes via formation of porous metal surfaces with the use of electroless deposition techniques. We propose the application of the developed device in direct analysis of neurotransmitters as well as comprehensive online coupling with microdialysis sampling in pharmacology, drug delivery, drug kinetics, and tissue metabolism studies.
Improving the performance of hypercrosslinked monolithic stationary phases and their application in separations of polar compounds
Czech Science Foundation, postdoctoral project P206/12/P049 (2012 – 2014).
The aim of this project is to prepare and optimize hypercrosslinked poly(styrene-vinylbenzylchloride-divinylbenzene) monolithic stationary phases. The internal structure of hypercrosslinked monoliths with relatively thin hypercrosslinked layer on the pore surface evokes the structure of superficially porous particles. The optimization of this porous layer can bring the properties of hypercrosslinked monoliths near the properties of currently available highly efficient columns packed with superficially porous particles. The tailored chemical modification of the reactive groups located at the pore surface is additionally proposed. This modification should enable the formation of libraries of new types of stationary phases that can be prepared from just a single optimized generic monolithic structure. The project proposes the preparation of stationary phases with functionalities suitable for hydrophilic interaction liquid chromatography. The results gained will provide the separation of complex samples containing polar compounds, such as drugs, metabolites, and other natural compounds.