Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA.
Methods Mol Biol. 2022;2500:31-42. doi: 10.1007/978-1-0716-2325-1_4.
Top-down proteomics methods have a distinct advantage over bottom-up methods in that they analyze intact proteins rather than digested peptides which can result in loss of information regarding the intact protein. However, the analysis of intact proteins using top-down proteomics methods has been impeded by the low resolution of typical separation approaches applied in bottom-up proteomics studies. To increase the coverage of intact proteomes, orthogonal, two-dimensional separation techniques have been developed to improve the separation efficiency; in this chapter, we describe a two-dimensional HPLC separation technique that utilizes a high-pH mobile phase in the first dimension followed by a low-pH mobile phase in the second dimension. This two-dimensional pH-based HPLC approach demonstrates increased separation efficiency of intact proteins and increased proteome coverage when compared to one-dimensional HPLC in the analysis of larger and lower abundance proteoforms.
自上而下的蛋白质组学方法相对于自下而上的方法具有明显的优势，因为它们分析的是完整的蛋白质，而不是消化后的肽，这可能导致与完整蛋白质有关的信息丢失。然而，使用自上而下的蛋白质组学方法分析完整的蛋白质受到典型的自下而上蛋白质组学研究中应用的分离方法分辨率低的阻碍。为了提高完整蛋白质组的覆盖率，已经开发了正交的二维分离技术来提高分离效率；在本章中，我们描述了一种二维 HPLC 分离技术，该技术在第一维中使用高 pH 流动相，然后在第二维中使用低 pH 流动相。与一维 HPLC 相比，这种基于 pH 的二维 HPLC 方法在分析较大和低丰度蛋白质时，提高了完整蛋白质的分离效率和蛋白质组的覆盖率。
