It is well known that protein unfolding during hydrophobic interaction chromatography (HIC) can lead to loss of product activity, irreversible binding, and poor selectivity. Less certain is the relationship between solution phase stability and how a protein unfolds on a HIC surface.  We utilize hydrogen-exchange mass spectrometry to investigate the conformation of multiple proteins adsorbed on HIC surfaces.  Multi-domain proteins known to unfold in solution via intermediates were adsorbed on HIC resins of varying hydrophobicity.  Exchange behavior at the domain level is investigated with proteolytic digestion to observe at what degree of HIC hydrophobicity individual domains unfold.  We observe that the order of domain thermodynamic stability in solution is important in determining what order domains will unfold as HIC surface hydrophobicity increases.  Comparisons of domain free energies of unfolding in solution and when adsorbed are made to quantify the effect individual HIC surfaces have on domain stability.

Comparison of unfolding pathways of proteins in solution and on hydrophobic surfaces as investigated by hydrogen-exchange mass spectrometry

243rd National Meeting (2012)

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