Abstract
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.
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