title: Proteome wide evaluation of the separation ability of hydrophobic interaction chromatography by fluorescent dye binding analysis.
abstract: Hydrophobic interaction chromatography (HIC) is an important tool in the industrial purification of proteins from various sources. The HIC separation behavior of individual (or model) proteins has been widely researched by others. On the contrary, this study focused on the fractionation ability of HIC when it is challenged with whole proteomes. The impact of the nature of three different proteomes, that is, yeast, soybean, and Chinese hamster ovary cells, on HIC separation was investigated. In doing so, chromatography fractions obtained under standardized conditions were evaluated in terms of their overall hydrophobicity--as measured by fluorescence dye binding. This technique allowed for the calculation of an average protein surface hydrophobicity (S(0)) for each fraction; a unique correlation between S(0) and the observed chromatographic behavior was established in each case. Following a similar strategy, the effect of three different ligands (polypropylene glycol, phenyl, and butyl) and two adsorbent particle sizes (65 and 100 µm) on the chromatographic behavior of the yeast proteome was evaluated. As expected, the superficial hydrophobicity of the proteins eluted is correlated with the salt concentration of its corresponding elution step. The findings reveled how--and in which extent--the type of ligand and the size of the beads actually influenced the fractionation of the complex biological mixture. Summarizing, the approach presented here can be instrumental to the study of the performance of chromatography adsorbents under conditions close to industrial practice and to the development of downstream processing strategies.
authors.affiliation: Downstream Bioprocessing Laboratory, School of Engineering and Science, Jacobs University, Campus Ring 1, Bremen, D-28759, Germany; Centro de Biotecnología-FEMSA, Departamento de Biotecnología e Ingeniería de Alimentos, Tecnológico de Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, 64849, Mexico.
authors.forename: Celeste C
authors.initials: CC
authors.lastname: Ibarra-Herrera
authors.forename: Rami
authors.initials: R
authors.lastname: Reddy-Vennapusa
authors.forename: Marco
authors.initials: M
authors.lastname: Rito-Palomares
authors.forename: Marcelo
authors.initials: M
authors.lastname: Fernández-Lahore
mesh_terms: D000818:Animals; D016466:CHO Cells; D002853:Chromatography, Liquid / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D020543:Proteome / Q000032:analysis*
chemical_list: D005456:Fluorescent Dyes; D020543:Proteome
keywords: adsorption; chromatography; hydrophobicity; ligand; particle size; proteome