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.
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