Thesis final - after defense-7 - Jacobs University

Chapter 3
3.2.3.2. The electrophoretic separation and proteins identification
The chromatographic fractions were further explored for their protein content by 2-D PAGE.
The protein concentrations of the chromatographic fractions were kept similar during their
electrophoretic separation by 2-D PAGE. This would give an insight to the total mass and the
number of protein contaminants recovered in each chromatographic fraction (19). The 2-D
gels were important for mapping the chromatographic separation of the yeast cell proteome
and to excise certain spots for protein identifications (Figures 21-23). The seventy seven
proteins were identified by MALDI-ToF-MS from all the gels representing specific
chromatographic fractions (Tables 12-14). The four proteins were visible on the gels with the
naked eyes, however were not visible when photographed and it is sometimes the case with
the weakly stained proteins (105-111). The proteins identified from each chromatographic
fraction were collected at different salt concentrations and can be separated through isocratic
chromatography. This will avoid trial and error chromatographies to figure out the elution
position of any of the tabulated protein. The large scale identification and characterization of
the proteins has explored the contaminant profile of the yeast cell proteome and is an addition
to the proteomics technology. The identified proteins and the corresponding 2-D gels can also
be used in future to identify proteins by relying on 2-D gel electrophoresis image databases
(123). The previous work on HIC was limited to a few homologous model proteins and
average surface hydrophobicity parameter, which may not portray a global picture of the
retention mechanism during HIC (23, 62). However, the global analysis of the base supports
and protein properties as a function of complex cell proteome has overwhelmed the
shortcomings in the previous approaches.
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