Thesis final - after defense-7 - Jacobs University

Chapter 3
3. Results and Discussion
3.1. Influence of the different ligand chemistries during hydrophobic
interaction chromatography
3.1.1. Summary
Several reports are available in literature in which various ligands have been compared based
on the retention time of model proteins in hydrophobic interaction chromatography (HIC) (62,
87, 99). However, the different ligands were comparatively investigated for the first time with
the proteome wide approach. A proteome wide approach has direct industrial applicability and
has several advantages over work performed with model proteins. This work is in the
continuation of a previous report from our group, where the ion exchange beads were used to
investigate the separation behavior of the insect cell proteome (19). Several conclusions were
made in that report, although the approach was not complete in its essence. The mentioned
approach was extended in this work for the comparative studies of the hydrophobic ligands
employing some additional steps of mass spectrometry and bioinformatics; which were
missing in the previous approach. This approach analyzed the chromatographic separation of
the overall protein contaminants in the host cell proteome instead of focussing on any of the
target protein. The Saccharomyces cerevisiae cell proteome was fractionated by different
ligands during HIC. The chromatographic fractions were collected at specific salt
concentrations, followed by their separation on two-dimensional polyacrylamide gel
electrophoresis (2-D PAGE). Several spots were selected from each 2-D gel and subjected to
identification by Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass
Spectrometry (MALDI-ToF-MS). Later on, several protein properties such as average surface
hydrophobicity, average hydrophobicity, average flexibility, average bulkiness and average
polarity were calculated for the identified proteins using different bioinformatics tools. These
properties were investigated for the first time with the proteome wide approach and correlated
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