Thesis final - after defense-7 - Jacobs University

Chapter 3
The applied protein load (40 mg) for chromatographic experiments was within the range of
dynamic binding capacities of the adsorbents. A high amount of proteins was adsorbed on
Toyopearl-Hexyl- and -Butyl which confirmed the high dynamic binding capacities of these
adsorbents. Toyopearl-Ether has shown less binding affinity and most of the proteins were
lost in flow through as non retained components. The results revealed the binding affinities of
the adsorbents for a cell proteome. In case of Toyopearl-Ether, almost 80% of the total load
was lost during washing as evidenced by the Bradford method. However, for Toyopearl-Butyl
and -Hexyl almost 50% of the proteins were lost in the flow through. The remaining half of
the proteins were retained in the column and eluted according to their hydrophobicity during
chromatography. A high amount of proteins was lost during flow through which confirmed
the less hydrophobic nature of the S. cerevisiae cell proteome (104). The results suggested the
use of high salt concentrations in order to increase the adsorption affinity of the yeast cell
proteome during HIC.
3.1.3.2. Two dimensional gel electrophoresis and proteins identification
The two dimensional gels were performed for all the chromatography fractions collected
within a specific operational window. Several proteins were selected from each gel for
identification purposes; however in a few cases the tiny and weakly stained proteins were
identified. Four out of the total identified proteins were visible on the gels with naked eyes,
however were not visible when photographed and it is sometimes the case with weakly
stained proteins (Figures 12-14). There are several reports where weakly stained proteins
were identified, but the spots were not visible on the 2-D gel pictures (105-111). In total,
ninety six proteins were identified using MALDI-ToF-MS, which revealed the applicability of
the in-gel digestion method optimized in this work specifically for the proteins fractionated by
HIC (Tables 5-7). The identified proteins can further provide the contaminant profile of the
yeast cell proteome and is an addition to the proteomics technology. In the case of Toyopearl-
47