Gospodarek Adrian M, Sun Weitong, O'Connell John P, Fernandez Erik J
Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904 4741, USA.
Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904 4741, USA.
J Chromatogr A. 2014 Dec 5;1371:204-19. doi: 10.1016/j.chroma.2014.10.080. Epub 2014 Oct 31.
In hydrophobic interaction chromatography (HIC), interactions between buried hydrophobic residues and HIC surfaces can cause conformational changes that interfere with separations and cause yield losses. This paper extends our previous investigations of protein unfolding in HIC chromatography by identifying protein structures on HIC surfaces under denaturing conditions and relating them to solution behavior. The thermal unfolding of three model multidomain proteins on three HIC surfaces of differing hydrophobicities was investigated with hydrogen exchange mass spectrometry (HXMS). The data were analyzed to obtain unfolding rates and Gibbs free energies for unfolding of adsorbed proteins. The melting temperatures of the proteins were lowered, but by different amounts, on the different surfaces. In addition, the structures of the proteins on the chromatographic surfaces were similar to the partially unfolded structures produced in the absence of a surface by temperature as well as by chemical denaturants. Finally, it was found that patterns of residue exposure to solvent on different surfaces at different temperatures can be largely superimposed. These findings suggest that protein unfolding on various HIC surfaces might be quantitatively related to protein unfolding in solution and that details of surface unfolding behavior might be generalized.
在疏水相互作用色谱法（HIC）中，埋藏的疏水残基与HIC表面之间的相互作用会导致构象变化，从而干扰分离并导致产量损失。本文通过鉴定变性条件下HIC表面上的蛋白质结构并将其与溶液行为相关联，扩展了我们先前对HIC色谱中蛋白质展开的研究。使用氢交换质谱（HXMS）研究了三种不同疏水性的HIC表面上三种模型多结构域蛋白质的热展开。分析数据以获得吸附蛋白质展开的速率和吉布斯自由能。在不同表面上，蛋白质的解链温度降低了，但降低的幅度不同。此外，色谱表面上蛋白质的结构类似于在没有表面的情况下通过温度以及化学变性剂产生的部分展开结构。最后，发现在不同温度下不同表面上残基暴露于溶剂的模式在很大程度上可以叠加。这些发现表明，蛋白质在各种HIC表面上的展开可能与溶液中蛋白质的展开在数量上相关，并且表面展开行为的细节可能具有普遍性。
