A proteome wide approach to understand chromatography behavior on hydrophobic adsorbents

  • The separation behavior of the Saccharomyces cerevisiae cell proteome was explored by hydrophobic interaction chromatography (HIC). Several beaded adsorbents were studied. A first group of HIC adsorbents (n = 3) harbored the same ligand moiety (i.e., Phenyl) but presented differences in the chemical nature of the matrix backbone. On the other hand, a second group of adsorbents (n = 3) presented the same chemical structure (i.e. based on polymethacrylate; Toyopearl / TP) but differed in the chemical nature of the HIC ligands. Chromatography runs were performed under typical conditions employing ammonium sulfate / phosphate buffer (pH = 7.5) as a mobile phase. Chromatography fractions were collected and further analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE); selected spots were identified by MALDI-ToF-MS and database search. A comparative analysis of protein separation behavior is presented as a function of the adsorbent type. As expected, TP-Hexyl and TP-Butyl showed increased protein retention in comparison with TP-Ether. Moreover, an influence of protein size and isoelectric point (pI) was also revealed. 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. Protein characteristics such as average surface hydrophobicity, average hydrophobicity average polarity, average bulkiness and average flexibility were tabulated for the identified spots and correlated with chromatography behavior. The proteins properties revealed limited ability to differentiate among the ligands and base supports for their hydrophobicity. This work -for the first time- studied the separation behavior of a real and complex cell proteome as a function of commercially available HIC adsorbents. The mentioned results will favor the understanding and the application of HIC, a method of choice in many industrial bio-separation schemes.

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:Nawab Ali
Referee:Marcelo Fernandez Lahore, Matthias Ullrich, Abid Riaz Ahmad
Advisor:Marcelo Fernandez Lahore
Persistent Identifier (URN):urn:nbn:de:101:1-201305294624
Document Type:PhD Thesis
Date of Successful Oral Defense:2012/06/05
Year of Completion:2012
Date of First Publication:2012/07/10
PhD Degree:Biochemical Engineering
School:SES School of Engineering and Science
Library of Congress Classification:T Technology / TP Chemical technology / TP155-156 Chemical engineering
Call No:Thesis 2012/15

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