Cabrera Rosa, Zhelyazkova Petya, Galvis Leonardo, Fernandez-Lahore Marcelo
Downstream Processing Laboratory, School of Engineering and Science, Jacobs University, Bremen, Germany.
J Sep Sci. 2008 Jul;31(13):2500-10. doi: 10.1002/jssc.200800076.
Surface charge, molecular weight, and folding state are known to influence protein chromatographic behaviour onto ion exchangers. Experimentally, information related to such factors can be gathered via 2-DE methods. The application of 2-D PAGE under denaturing/reducing conditions was already shown to reveal separation trends within a large protein population from cell extracts. However, ion-exchange chromatography normally runs under native conditions. A tailored protocol consisting in a first separation based on IEF on Immobiline strips under native conditions followed by a second dimension SDS-PAGE run was adopted. The chromatographic versus electrophoretic separation behaviours of two model proteins, thaumatin (TAU) and BSA, were compared to better understand which proteomic routine would be better suited to anticipate IEX chromatographic separations. It was observed that the information contained in the pI value obtained with the adapted 2-DE protocol showed better correlation with the IEX chromatographic behaviour. On the other hand, chromatographic separations performed in the presence of urea as a denaturant have demonstrated the potential influence of hydrodynamic radius/conformation on protein separation. Moreover, the information provided by such 2-D system correlated well with the chromatographic behaviour of an additional set of pure proteins. An initial prediction of protein ion-exchange chromatographic behaviour could be possible utilizing an experimental approach based on 2-DE running under milder chemical conditions. This technique provides information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock.
已知表面电荷、分子量和折叠状态会影响蛋白质在离子交换剂上的色谱行为。通过实验，可以借助二维电泳（2-DE）方法收集与这些因素相关的信息。已证明在变性/还原条件下进行二维聚丙烯酰胺凝胶电泳（2-D PAGE）能够揭示细胞提取物中大量蛋白质群体的分离趋势。然而，离子交换色谱通常在天然条件下运行。我们采用了一种定制方案，首先在天然条件下基于固定化pH梯度（IEF）在固相pH梯度胶条上进行分离，然后进行第二维的十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）。比较了两种模型蛋白，奇异果甜蛋白（TAU）和牛血清白蛋白（BSA）的色谱分离行为与电泳分离行为，以更好地了解哪种蛋白质组学方法更适合预测离子交换色谱（IEX）分离。结果发现，采用改进的二维电泳方案获得的等电点（pI）值所包含的信息与离子交换色谱行为具有更好的相关性。另一方面，在有尿素作为变性剂的情况下进行的色谱分离表明了流体动力学半径/构象对蛋白质分离的潜在影响。此外，这种二维系统提供的信息与另一组纯蛋白的色谱行为也有很好的相关性。利用基于在较温和化学条件下进行二维电泳的实验方法，有可能对蛋白质的离子交换色谱行为进行初步预测。该技术提供的信息与在复杂生物技术原料中观察到的分离行为更为相似。
