RPLC-RPLC-MS/MS for Proteoform Identification.
RPLC-RPLC-MS/MS for Proteoform Identification.
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DOI：
10.1007/978-1-0716-2325-1_4
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发表时间：
2022
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自上而下的蛋白质组学方法与自下而上的方法相比具有明显的优势，因为它们分析完整的蛋白质而不是消化的肽，消化的肽可能导致关于完整蛋白质的信息丢失。然而，使用自上而下的蛋白质组学方法分析完整蛋白质一直受到自下而上的蛋白质组学研究中应用的典型分离方法的低分辨率的阻碍。为了增加完整的蛋白质组的覆盖率，正交，二维分离技术已经开发出来，以提高分离效率，在本章中，我们描述了一个二维HPLC分离技术，利用高pH值的移动的相在第一维，其次是低pH值的移动的相在第二维。与一维HPLC相比，这种基于二维pH的HPLC方法证明了完整蛋白质的分离效率增加，并且蛋白质组覆盖率增加，以分析更大和更低丰度的蛋白质型。
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 the proteome coverage when compared to one-dimensional HPLC to analyze larger and lower abundance proteoforms.