Peptit ve Protein-PEG Konjugatlarının Sentezi ve Kütle Spektrometrik Analizi

Abstract

Therapeutic proteins are widely used in the field of pharmacology, and there are many clinical applications in this area that use hybrid molecules containing therapeutic protein structures. With polyethylene glycol (PEG), not only the therapeutic proteins, but also other therapeutic biomolecules and drugs, the conjugation process called pegylation is performed and hybrid molecules are formed. One of the biggest challenges encountered in these studies is the analysis of the molecules obtained as a result of the pegylation process, the determining of the binding sites and their binding rates correctly. Analysis and characterization of these polymer-based therapeutic hybrid molecules is analytically quite tough.

In this thesis, it is aimed to develop analytical methods which are easier and do not take time for the analysis and characterization of these hybrid molecules by synthesizing hybrid peptide-polymer and protein-polymer molecules. For this purpose, human angiotensin II and human insulin molecules have been used as exemplary polypeptide structures which are intended to be conjugated with PEG. For the conjugation reaction, PEG samples were used at different molecular weights distribution. The end groups of some of the PEG samples used were derivatized into different reactive groups. The conjugation reactions were carried out under both organic and physiological conditions. Mass spectrometric analyzes of the hybrid molecules, ie conjugates were performed by using the MALDI-MS system. The MALDI-MS / MS method was used to determine the biomolecule binding sites and binding rates of PEG chains. The bottom-up proteomic analysis approach was carried out for the characterization of large structure hybrid molecules containing insulin and then the binding regions and binding rates of PEG chains were determined by the MALDI-MS / MS method. As a result of this thesis, PEG chains’ end groups was modified differently and caried out successfully the conjugation reactions in which used these PEG chains have been determined by using MALDI-MS method. The determination of the site and ratio of the PEG chains on the biomolecule was carried out in a practical and rapid manner by using sample preparation and MALDI-MS / MS analysis methods developed within the scope of the thesis.