Xiu Lichen, Valeja Santosh G, Alpert Andrew J, Jin Song, Ge Ying
Department of Chemistry, University of Wisconsin-Madison , Madison 53706, Wisconsin, United States.
Anal Chem. 2014 Aug 5;86(15):7899-906. doi: 10.1021/ac501836k. Epub 2014 Jul 9.
One of the challenges in proteomics is the proteome's complexity, which necessitates the fractionation of proteins prior to the mass spectrometry (MS) analysis. Despite recent advances in top-down proteomics, separation of intact proteins remains challenging. Hydrophobic interaction chromatography (HIC) appears to be a promising method that provides high-resolution separation of intact proteins, but unfortunately the salts conventionally used for HIC are incompatible with MS. In this study, we have identified ammonium tartrate as a MS-compatible salt for HIC with comparable separation performance as the conventionally used ammonium sulfate. Furthermore, we found that the selectivity obtained with ammonium tartrate in the HIC mobile phases is orthogonal to that of reverse phase chromatography (RPC). By coupling HIC and RPC as a novel two-dimensional chromatographic method, we have achieved effective high-resolution intact protein separation as demonstrated with standard protein mixtures and a complex cell lysate. Subsequently, the separated intact proteins were identified by high-resolution top-down MS. For the first time, these results have shown the high potential of HIC as a high-resolution protein separation method for top-down proteomics.
蛋白质组学面临的挑战之一是蛋白质组的复杂性，这使得在进行质谱（MS）分析之前必须对蛋白质进行分级分离。尽管自上而下的蛋白质组学最近取得了进展，但完整蛋白质的分离仍然具有挑战性。疏水相互作用色谱法（HIC）似乎是一种很有前景的方法，可实现完整蛋白质的高分辨率分离，但不幸的是，传统上用于HIC的盐与MS不兼容。在本研究中，我们已确定酒石酸铵是一种与MS兼容的HIC盐，其分离性能与传统使用的硫酸铵相当。此外，我们发现酒石酸铵在HIC流动相中获得的选择性与反相色谱法（RPC）的选择性正交。通过将HIC和RPC耦合作为一种新型二维色谱方法，我们已实现了有效的高分辨率完整蛋白质分离，标准蛋白质混合物和复杂细胞裂解物的实验结果证明了这一点。随后，通过高分辨率自上而下的MS鉴定了分离出的完整蛋白质。这些结果首次表明HIC作为自上而下蛋白质组学的高分辨率蛋白质分离方法具有很高的潜力。
