New fractionation tools targeting elusive post-translational modifications

Protein phosphorylation is a reversible post-translational modification (PTM)playing a central role in numerous biological events including disease pathogenesis.Thus, the analysis of phosphoproteome is crucial for understandingcellular regulation processes and can facilitate the development of new diagnosticand therapeutic tools.Phosphoproteins are typically analyzed using liquid chromatography coupledwith mass spectrometry (LC-MS) after proteolytic processing. However,phosphopeptides are notoriously difficult to analyze by LC-MS due their lowabundance and transient nature. This creates a need for effective enrichmenttools for phosphorylated proteins and peptides prior to mass spectrometryanalysis.The work presented in this thesis is focused on development and validationof methods and tools for enrichment of phosphopeptides with the use of molecularimprinting technology. In particular, the targeted PTMs include phosphorylationon tyrosine (pTyr) and histidine (pHis).The key recognition element employed in developed synthetic receptors was1,3-diaryl urea functional monomer FM1. This monomer is a potent hydrogenbond donor forming strong cyclic hydrogen bonds with oxyanions such asphosphates. The bias of the imprinted urea-based receptor towards differentphosphorylated residues can be programmed by selection of the template. Thus, the N, C-protected phosphotyrosine and phosphonotriazolylalaninewere used as templates to generate phosphotyrosine (pTyr MIP) and phosphohistidine(pHis MIP) selective molecularly imprinted polymers, respectively.The application of previously reported pTyr MIP for phosphoproteomicstudies was validated on complex biological samples of the mouse brain lysatedigest spiked with standard peptides and HeLa cells digested proteins. Furthermore,the pTyr MIP was developed in the format of microspherical porous beads characterized by uniformly sized and shaped particles with increasedsurface area and pore size as well as improved binding affinity and sel