Thesis final - after defense-7 - Jacobs University

Chapter 3
3.3.2.2. Protein distributions on 2-D gels
Proteins derived from S. cerevisiae cell proteome have shown less binding affinity on the
adsorbents based on hydrophobic interactions. A maximum number and a high amount (mg)
of proteins were eluted at the beginning of the chromatography than at the end (Figure 29 A-
B). These results confirmed the less hydrophobic nature of S. cerevisiae cell proteome. The
hydrophobic proteins in the S. cerevisiae cell proteome were reported to be 16% of the total
cell proteome. This was very less amount than reported for other cell proteomes (104). The
less hydrophobic nature of the yeast cell proteome suggested high hydrophobic conditions for
the efficient separation of the cell proteome during HIC. The analysis of 2-D gels has also
revealed the variation in the binding affinity of the different ligands for the same proteome. In
case of Toyopearl-Ether, a higher number of protein spots were eluted at the beginning of the
chromatography than at the end. The reason could be the less hydrophobic nature of the
Toyopearl-Ether. In contrast, Toyopearl-Hexyl has shown higher numbers of protein spots at
the end of the chromatography than at the beginning because of its high hydrophobic nature.
In the similar fashion, the 2-D gels analysis has also revealed the variation in the binding
affinity of the base supports for the same proteome. In case of Toyopearl-Phenyl, a higher
number of proteins were eluted in the beginning of the chromatography than at the end and
the reason could be the less hydrophobic nature of the base support. Sepharose-Phenyl and
Source-Phenyl have shown higher numbers of spots at the end of the chromatography. This
revealed the high binding affinity of the two adsorbents for the yeast cell proteome.
When the adsorption affinity of polymethacrylate based media was compared with phenyl
ligated media, the adsorption affinity was higher in earlier than later one. Toyopearl-Phenyl
with phenyl ligand has not given higher adsorption of proteins when compared to Toyopearl-
Butyl and Toyopearl-Hexyl. This confirmed that aromatic interactions have no effect on the
adsorption affinity of the adsorbents (122, 132).
104