New fractionation tools targeting elusive post-translational modifications

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New fractionation tools targeting elusive post-translational modifications

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dc.contributor.author Wierzbicka, Celina
dc.date.accessioned 2017-03-22T11:03:41Z
dc.date.available 2017-03-22T11:03:41Z
dc.date.issued 2017 en_US
dc.identifier.isbn 9789171047281 en_US
dc.identifier.isbn 9789171047298 en_US
dc.identifier.issn 1653-5383 en_US
dc.identifier.uri http://hdl.handle.net/2043/22413
dc.description.abstract 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 understanding cellular regulation processes and can facilitate the development of new diagnostic and therapeutic tools. Phosphoproteins are typically analyzed using liquid chromatography coupled with mass spectrometry (LC-MS) after proteolytic processing. However, phosphopeptides are notoriously difficult to analyze by LC-MS due their low abundance and transient nature. This creates a need for effective enrichment tools for phosphorylated proteins and peptides prior to mass spectrometry analysis. The work presented in this thesis is focused on development and validation of methods and tools for enrichment of phosphopeptides with the use of molecular imprinting technology. In particular, the targeted PTMs include phosphorylation on tyrosine (pTyr) and histidine (pHis). The key recognition element employed in developed synthetic receptors was 1,3-diaryl urea functional monomer FM1. This monomer is a potent hydrogen bond donor forming strong cyclic hydrogen bonds with oxyanions such as phosphates. The bias of the imprinted urea-based receptor towards different phosphorylated residues can be programmed by selection of the template. Thus, the N, C-protected phosphotyrosine and phosphonotriazolylalanine were 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 phosphoproteomic studies was validated on complex biological samples of the mouse brain lysate digest 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 increased surface area and pore size as well as improved binding affinity and selectivity for larger pTyr peptides (2-3 kDa). This opens the way to generation of capture materials suitable for middle-down phosphoproteomics. In response to the lack of adequate tools and methods for enrichment of acid- labile phosphohistidine peptides a pHis MIP-based approach is proposed as a solution. The method involving selective dephosphorylation of phosphoserine (pSer) peptide by alkali treatment of the sample, followed by extraction of base-stable pHis peptides with MIP was demonstrated on the sample of bovine serum albumin digest spiked with standard pSer and pHis peptides. The last part of this thesis is focused on improving the recognition of phosphopeptides in aqueous media – the natural environment of biological samples. Guided by the principles of supramolecular chemistry, novel cationic host monomers were introduced for binding phosphates by ionic hydrogen bonds. These were used to synthesize MIPs showing enhanced binding of phosphopeptides in aqueous media. en_US
dc.format.extent 63
dc.language.iso eng en_US
dc.publisher Malmö university, Faculty of Health and Society en_US
dc.relation.ispartofseries Malmö University Health and Society Dissertations;3 sv
dc.relation.haspart I. Chen, J.; Shinde, S.; Subedi, P.; Wierzbicka, C.; Sellergren, B.; Helling, S.; Marcus, K. Validation of molecularly imprinted polymers for side chain selective phosphopeptide enrichment. Journal of Chromatography A, 1471 (2016) 45–50. en_US
dc.relation.haspart II. Bllaci, L.; Torsetnes, S. B.; Wierzbicka, C.; Shinde, S.; Sellergren, B.; Rogowska-Wrzesinska, A.; Jensen, O. N. Phosphotyrosine biased enrichment of tryptic peptides from cancer cells exploiting combined pY MIP and TiO2 affinity. Manuscript. en_US
dc.relation.haspart III. Wierzbicka, C.; Torsetnes, S. B.; Jensen, O. N.; Shinde, S.; Sellergren, B. Hierarchically templated beads with tailored pore structure for phosphopeptide capture and phosphoproteomics. RSC Advances, Accepted (2017). en_US
dc.relation.haspart IV. Wierzbicka, C.; Gajoch, K.; Jensen, O. N.; Sellergren, B. Selective enrichment of histidine phosphorylated peptides combining β-elimination and MIP-based pHis capture. Submitted for publication. en_US
dc.relation.haspart V. Wierzbicka, C.; Liu, M.; Irgum, K.; Sellergren, B. Cationic pTyr/pSer imprinted polymers based on a bis-imidazolium host monomer: Phosphopeptide recognition in aqueous buffers demonstrated by μ- liquid chromatography and monolithic columns. Journal of Materials Chemistry B, 5 (2017) 953-960. en_US
dc.subject Molecular imprinting en_US
dc.subject Molecular recognition en_US
dc.subject Phosphopeptides en_US
dc.subject Proteomics en_US
dc.subject Phosphotyrosine en_US
dc.subject Phosphohistidine en_US
dc.subject.classification Medicine en_US
dc.title New fractionation tools targeting elusive post-translational modifications en_US
dc.type Doctoral Thesis
dc.identifier.paperprint 0 en_US
dc.contributor.department Malmö University. Faculty of Health and Society en_US
dc.contributor.department Malmö University. Biomedical Sciences (BMV) en_US
dc.identifier.doi 10.24834/2043/22413
dc.subject.srsc Research Subject Categories::ODONTOLOGY en_US
dcterms.type Doctoral Thesis, comprehensive summary
mahlocal.rights.eplikt Yes
mahlocal.rights.oaType green
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