Kütle Spektrometrisi İle Proteinlerin Ubikitinasyon Bölgelerinin Düşük Derişimlerde Belirlenmesi
Özet
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
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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.
