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Identifier	000442932
Title	Ανάπτυξη νέων αναλυτικών τεχνικών υγρής χρωματογραφίας συζευγμένης με φασματομετρία μάζας επαγωγικά συζευγμένου πλάσματος για την μεταλλοπρωτεωμική ανάλυση περιβαλλοντικών και βιολογικών δειγμάτων
Alternative Title	Development of new analytical techniques of liquid chromatography coupled with inductively coupled plasma mass spectrometry for the metalloproteomic analysis of environmental and biological samples
Author	Πανάγου, Γεωργία Κ.
Thesis advisor	Περγαντής, Σπυρίδων
Reviewer	Λυδάκης - Σημαντήρης, Νικόλαος Μήλιος, Κωνσταντίνος
Abstract	Metalloproteins make up almost one third of all proteins in biological systems. Their role is crucial for numerous biological functions, since the majority of them are enzymes contributing to the homeostatic mechanisms of metals. Metalloproteomics is a useful tool for the structural and functional characterization of metalloproteins, while also making their quantification possible. These characterizations contribute to the better understanding of numerous processes that occur in biological systems. Liquid chromatography techniques coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS), have been widely used, in the past few decades, for the metalloproteins analysis due to their numerous advantages. More specifically, size exclusion chromatography and affinity chromatography enable the separation of metalloproteins in mild conditions, which prevents the denaturation of the proteins and therefore the loss of metal content. Moreover, ICP-MS allows for multielemental analysis in complicated biologic matrices, characterized by high sensitivity. This thesis consists of the development of a technique that combines size exclusion chromatography (HiTrap Desalting HP) and affinity chromatography (HiTrap Blue-HP) coupled online with inductively coupled plasma mass spectrometry (ICP-MS) for the analysis of metalloproteins which contain Mg, Ca, Se, Fe, Co, Ni or/and Ζn in human blood serum samples. In these analyses, the use of universal cell as a collision cell (He, 2 ml/min) has an advantage over its use as a reaction cell (O2, 1.5 ml/min). From the analysis of human blood serum samples of different athletes, for the majority of metals, two chromatographic profiles emerge. In samples of the same athlete, that had been taken before and after the exercising, are changes have been mainly observed in the concentration of metals, which indicates that exercising affects the form and concentration of metals in human blood serum. Moreover, each metal fraction has been matched with metalloproteins found in human serum, in accordance with bibliography. In addition, this thesis includes the development of a size exclusion chromatography technique coupled with inductively coupled plasma mass spectrometry for the rapid analysis of selenoproteins in Chlamydomonas reinhardtii cells. This technique is used for the study of different selenium forms in cells that have been cultivated in the presence of different concentrations of SeO4-. The chromatographs, of the lysates of each cell culture, indicate the accumulation of selenium by the cells. Part of the accumulated Se is incorporated in macromolecules (>5 kDa), until the concentration of Se(VI) exceeds 100 μM. The rest of Se(VI) quantity can be found in the cells in inorganic or low molecular weight organic forms (<1 kDa). Comparing the total concentration of Se, measured with SEC-ICP-MS, with the respective values from Conventional ICP-MS analysis, a high recovery of >96% is obtained.
Language	Greek
Issue date	2021-11-24
Collection	School/Department--School of Sciences and Engineering--Department of Chemistry--Post-graduate theses
	Type of Work--Post-graduate theses
Permanent Link	https://elocus.lib.uoc.gr//dlib/4/6/1/metadata-dlib-1634298124-777084-16490.tkl
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