Capillary zone electrophoresis-high field asymmetric ion mobility spectrometry-tandem mass spectrometry for top-down characterization of histone proteoforms

Characterization of histone proteoforms with various post-translational modifications (PTMs) is critical for a better understanding of functions of histone proteoforms in epigenetic control of gene expression. Mass spectrometry (MS)-based top-down proteomics (TDP) is a valuable approach for delineating histone proteoforms because it can provide us with a bird's-eye view of histone proteoforms carrying diverse combinations of PTMs. Here, we present the first example of coupling capillary zone electrophoresis (CZE), ion mobility spectrometry (IMS), and MS for online multi-dimensional separations of histone proteoforms. Our CZE-high-field asymmetric waveform IMS (FAIMS)-MS/MS platform identified 366 (ProSight PD) and 602 (TopPIC) histone proteoforms from a commercial calf histone sample using a low microgram amount of histone sample as the starting material. CZE-FAIMS-MS/MS improved the number of histone proteoform identifications by about 3 folds compared to CZE-MS/MS alone (without FAIMS). The results indicate that CZE-FAIMS-MS/MS could be a useful tool for comprehensive characterization of histone proteoforms with high sensitivity.

Automated summary

The characterization of histone proteoforms with various post-translational modifications is crucial for understanding their role in epigenetic control of gene expression. This study presents a novel approach combining capillary zone electrophoresis, ion mobility spectrometry, and mass spectrometry for online multi-dimensional separations of histone proteoforms. The results demonstrate that this technique can greatly improve the identification of histone proteoforms and provide a high sensitivity tool for their comprehensive characterization.