Kütle Spektrometrisi Uygulamalarında Fosfopeptitlerin Hassas Tayini İçin Yeni Stratejilerin Geliştirilmesi

Abstract

Most of the proteins may at some time be phosphorylated on serine, threonine or tyrosine aminoacids when they perform a certain function. Phosphoprotein is protein that contain phosphate group in structure. The smaller amino acid sequences found in the phosphate group are called phosphopeptides. Many vital biological functions can be explained by post translational modifications (PTMs), such as phosphorylation and dephosphorylation of proteins. During these modifications that occur in the hydroxyl groups of serine (Ser), threonine (Thr) and tyrosine (Tyr) amino acids, kinase enzymes bind phosphate groups to amino acids while phosphatase enzymes block phosphate groups. The role of many post-translational modifications in cellular activity, response, and development mechanisms remains unclear. Therefore, it is important to examine post-translational modifications and clarify their mechanisms.

In this thesis, phosphopeptides in peptide mixtures are enriched by sol-gels which are containing cationic moieties, expected to show affinity for phosphopeptides. Sol-gel materials were synthesized predominantly by using C6H6MgO7 (Magnesium citrate) salt and TEOS (tetraethylorthosilicate) chemical components. Magnesium is the fourth most abundant mineral in metabolism and plays a role in 300 different reactions within the metabolism. In addition to its role in activities such as protein synthesis, the Mg2+ cation, which is a hard acid according to Pearson's hard-soft acid and bases theory is expected to interact more strongly with a hard base, PO-4 anion. For this reason, different types of sol-gel materials which containing magnesium ions were synthesized. The C6H6MgO7-H3PO4@TEOS sol-gel, which was synthesized using H3PO4 (phosphoric acid) showed the highest performance in phosphopeptide enrichment studies. In the characterization studies

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carried out by ATR-FTIR (Attenuated Total Reflection-Fourier Transform Infrared) spectroscopy and TGA (Thermo Gravimetric Analysis), The C6H6MgO7-H3PO4@TEOS sol-gel structure contained more water than the other sol-gel structures and increased adsorption capacity of phosphopeptides by accelerating the diffusion rate into the sol-gel structure. Thus, the phosphopeptides at a concentration of 13 pmol/mL (13 fmol/μL) were successfully enriched from the solution. The surfaces of the sol-gel materials analyzed by XPS (X-Ray Photoelectron spectroscopy) technique and it was observed that TEOS (tetraethylorthosilicate), and Magnesium, which is effective for the successful enrichment of phosphopeptides, embedded into the structure. ESI mass spectra, obtained after the phosphopeptide enrichment studies, were investigated by open-source mMass software (www.mmass.org), and phosphopeptide enrichment rates were mathematically and scientifically demonstrated.