Thesis final - after defense-7 - Jacobs University

Chapter 4
• The base supports revealed less difference at high salt concentrations than at low salt
concentrations. In other words, the differences among the base supports were less at
high hydrophobic conditions than at low hydrophobic conditions. The reason could be
the effects of mobile phase hydrophobic conditions.
4.4. Effect of protein properties on separation behavior during chromatography
• Several protein properties were investigated for the first time with the proteome wide
approach for their effects on the protein retention behavior during HIC.
• The average surface hydrophobicity (ASH), average hydrophobicity (AH) and average
flexibility (AF) have revealed direct relationships with the retention behavior of the
proteins in HIC.
• The average bulkiness of the proteins did not show any relationship with the retention
behavior during chromatography.
• The polarity of the proteins exhibited an inverse relationship with the retention of the
proteins. The higher the polarity of the protein, the less time it will stay on the
adsorbent during chromatography and vice versa.
• An indirect relationship was observed between polarity and hydrophobicity; proteins
with high polarity were less hydrophobic in nature and vice versa.
• An indirect relationship was also observed between polarity and flexibility; proteins
with high polarity were less flexible in nature and vice versa.
• Among all the properties, ASH was the only property which has a decisive role in the
retention behavior of the proteins during HIC.
• The salt and ASH ranges were defined for the first time during the chromatographic
fractionation of the yeast cell proteome. The information gathered about ASH and the
retention position during chromatography can be exploited for the chromatographic
separation of recombinant proteins from host cell proteomes.
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