Evaluating C4 and Diphenyl Nanocapillary Columns for Top‐Down Analysis of Endogenous Histone Proteoforms and Larger Proteins

Top-down proteomics (TDP) enables the characterization of histone proteoforms. However, the extensive post-translational modifications and high sequence homology of these proteins pose significant separation challenges. Here, we compare C4 and diphenyl reversed-phase columns for top-down analysis of histone proteoforms. The TDP-based methods were developed using a mixture of six intact protein standards (~9-68 kDa) and applied to a cellular histone extract. While the C4 stationary phase favored the retention of more hydrophobic proteoforms, the diphenyl phase enhanced the separation of more polar species. In total, 55 histone proteoforms were identified, including 14 unique to C4 and 19 unique to diphenyl. These results demonstrate the orthogonal selectivity of the two stationary phases and highlight the importance of column chemistry in improving proteoform resolution in TDP workflows.