Süt Whey Proteinlerinin Kütle Spektrometrik Yöntemler N-Glikan Profillerinin Belirlenmesi
Özet
Post-translational modifications control many biological activities and it is known that about 300 different post-translational modifications occur in protein structures. Glycosylation is one of the most common forms of post-translational modifications of proteins. Glycans added to proteins as a result of glycosylation have important roles such as modification of immunological response, recognition, targeting and folding of proteins. It is important to understand the function of glycan diversity in cellular activities. Glycans play a critical role in intracellular interaction mechanisms. Particularly in the immune system, antibodies have critical functions in recognizing antigens.
Milk, a complex biological matrix containing nutritional components necessary for the healthy growth of newborns, has a natural protective function. The important functions of milk such as antibacterial, antimicrobial and contributing to the immune system have been attributed to whey proteins. Post-translational modifications of milk proteins not only expanded the complexity of their composition, but also contributed to their biological function. N-glycan profiles of milk whey proteins are important in order to better understand the biological functions of milk and to be used both for therapeutic purposes and to determine the type of milk that can replace breast milk. Little is known about glycans, especially their biological properties, which are associated with milk glycoproteins. Therefore, glycosylation analysis of milk whey proteins should be performed with reliable techniques. In this thesis, it is aimed to make N-glycan analysis of 5 different milk types including cow, goat, donkey, buffalo and breast milk. For the analysis of N-glycans, labeling with procainamide was chosen because of its advantage in mass spectrometric devices. In this process, all analyzes were carried out using fluorescence and mass spectrometric detectors based on hydrophilic interaction chromatography. As a result of the analyzes, glycan sequences of each species were determined. For human, cow, goat, buffalo and donkey, 27, 24, 30, 20 and 29 N-glycan structures were determined at MS / MS level, respectively. The relative abundance of N-glycan structures of each species was determined and comparisons were made between the species. Furthermore, glycan structures were grouped according to their common properties and further comparisons were made. H6N2, H4N4F1, H5N4F1, H5N4S1, H5N4F1S1 and H5N4F1S2 glycans were found to be common in all milk. According to the relative abundance of sialylated, N-glycan types containing the most sialic acid were found in goat milk. When it was evaluated in terms of fucose, it was determined that goats milk whey glycomes were highly fucosylated. It was determined that the distribution was followed by human, buffalo, cow and donkey species, respectively. N-glycan structures containing O-acetylated sialic acid were determined only in donkey milk. With this study, glycan analyzes of donkey and buffalo milk were made for the first time and all species were compared for the first time using the same method and device. A strong bioanalytical method has been developed using procainamide labeling and the HILIC-FLD-QTOF-MS/MS method to determine the N-glycan structure of milk proteins.
