Kütle Spektrometrisi İle Proteinlerin Ubikitinasyon Bölgelerinin Düşük Derişimlerde Belirlenmesi

Abstract

Post-translational modifications of proteins, such as phosphorylation, glycosylation, acetylation, ubiquitination and the other various post-translational modifications allow the regulation of many cellular functions and intracellular signaling events. Ubiquitination is a very common post-translational modification that plays a key role in the regulation and degradation of many protein groups. Analysis of ubiquitination and polyubiquitination is one of the most active study area in the field of proteomics. Ubiquitination is actively involved in the onset and progression of diseases such as cancer, metabolic syndromes, neurodegenerative diseases (Alzheimer, Parkinson, Huntington), inflammatory disorders, autoimmunity (the formation of antibodies against their own antigens in tissues), infection and muscular dystrophy. Identification of ubiquitination sites and enlightenment of the binding mechanisms for understanding the functions of ubiquitinated proteins are crucial in the development of diagnostic and therapeutic methods for diseases. Since the level of posttranslationally modified ubiquitinated proteins are very low in cells compared to their native forms, the detection of ubiquitination and ubiquitination sites is quite difficult. Analyzes needed for high sensitivity for the determination of ubiquitination sites are performed by using mass spectrometric techniques, where the most accurate and reliable results can be obtained in analyzes following a suitable enrichment method iv prior to the mass spectrometric analyses. In the scope of this thesis, based on the Histon 2B peptide and ubiquitinated Histon 2B peptide, basic analysis of the study was performed by optimizing various sample preparation and instrumental analysis parameters using Matrix Assisted Laser Desorption Ionization Time-of Flight Mass Spectrometry (MALDI-TOF-MS). As a result of the analyses, it was determined that it is possible to observe the ubiquitination as a post-translational modification mass spectrometrically by interpreting the peptide signals in the spectra of the reference biomolecules. In the further studies, an E3 ubiquitin protein ligase UHRF1 (Ubiquitinlike, containing PHD and RING finger domains, 1) was selected as an ubiquitinating enzyme to elucidate the role of ubiquitination in cancer cell death. The downregulation of UHRF1 in UHRF1_siRNA transfected cells proved that UHRF1_ siRNAs are specific to UHRF1 according to data analysis results of simultaneous proteomics studies on UHRF1_siRNA transfected RKO cells and control RKO cells. A significant increase in the number of detected ubiquitinated proteins was observed after the enrichment studies performed with the immunoaffinity precipitation. Therefore, it was decided that an efficient enrichment method for ubiquitination detection assay is required for the trypsin digested peptides. In the last part of the thesis, the poly-L-Lysine immobilized sol-gel, poly-L-arginine immobilized sol-gel, poly-L-ornithine immobilized sol-gel, titanium, zirconium based sol-gels, sulfonyl group containing surface, silk and finally, the cotton materials were incubated with the digestion products of the monoubiquitinated Histone 2B peptide. As a result of this enrichment experiments, it was found that the material with the best signal of ubiquitination region was sulfonyl group containing anionic surface material and could be used for the enrichment of ubiquitinated peptides in advanced studies.