Thesis final - after defense-7 - Jacobs University

Chapter 3
3.3. Protein distributions on 2-D gels: the influence of molecular
weight and isoelectric point coordinates
3.3.1. Summary
The separation behavior of the yeast cell proteome by hydrophobic interaction
chromatography (HIC) was examined utilizing two dimensional polyacrylamide gel
electrophoresis (2-D PAGE). The yeast cell proteome was fractionated on different
hydrophobic adsorbents and chromatographic fractions were collected on specific salt
concentrations. The chromatographic fractions were further analyzed by 2-D PAGE to
investigate the contaminant profile in each chromatographic fraction. This chapter will give
an additional understanding to the influence of ligands and base supports on the
chromatographic separation of the yeast cell proteome. As expected, Toyopearl-Hexyl and
Toyopearl-Butyl have shown increased protein retention in comparison with Toyopearl-Ether.
Moreover, an influence of protein size and isoelectric point (pI) was also revealed as a
function of the adsorbent type. Larger and / or neutral proteins showed increased while acidic
(pI < 6) and basic (pI > 8) proteins depicted decreased or increased retention, depending on
the adsorbent type.
In addition to the separation behavior of yeast cell proteome during chromatography, an
overview of the yeast cell proteome was gained. A high amount and a higher number of the
proteins on 2-D gels were reported at the beginning of the chromatography than at the end.
This confirmed the less hydrophobic nature of the yeast cell proteome. A higher number of
smaller and basic proteins were observed on 2-D gels in comparison to larger and acidic
proteins. This further showed the nature of the yeast cell proteome.
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