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Chapter 3
3.2.3. Results and Discussion
3.2.3.1. Chromatographic fractionation
Scouting experiments were performed to explore the most suitable operational conditions for
the chromatographic separations of the yeast cell proteome. These standardized
chromatographic conditions are summarized in Table 11. A decreasing gradient of
ammonium sulfate was performed from 1.6 to 0 M and seven fractions were collected at
specific salt concentrations in order to investigate the proteins available in each
chromatographic fraction. The chromatograms in Figure 20 depict the resolution behavior of
the three base supports for the same crude extract. There was no clear matching in the
separation profiles of the three adsorbents due to the differences in their base support
chemistries. The peaks with the number of 6, 4 and 1 correspond to Sepharose-Phenyl,
Source-Phenyl and Toyopearl-Phenyl, respectively. A high resolution was observed in case of
Sepharose-Phenyl and Source-Phenyl, due to their smaller bead size than Toyopearl-Phenyl.
One large peak was observed for Toyopearl-Phenyl, the reason could be the larger bead size
of the base support, which resulted in a low resolution during chromatographic separation.
The overall resolution of the three chromatograms was not high. The reason could be the high
amount of the sample load applied during chromatographic fractionation. In HIC, it is the
initial effort to report the selectivity of phenyl media for a crude extract. The experimental
approach applied in this work, where fractionation of a soluble cell proteome was done
instead of model proteins will pave the way for a direct comparison between adsorbents in
terms of hydrophobicity towards proteins derived from a complex biological mixture.
Information of this kind of work has been rarely available in literature where they utilized a
host cell proteome for chromatographic fractionation instead of the model proteins (63).
A high amount of proteins (almost 50%) was lost in the flow through and the remaining
proteins were retained in the column. This kind of behavior was also reported in Section 3.1,
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