Combining SDS‐PAGE to capillary zone electrophoresis‐tandem mass spectrometry for high‐resolution top‐down proteomics analysis of intact histone proteoforms

Abstract

Mass spectrometry (MS)-based top-down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post-translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high-resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS-PAGE-based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI-MS) with capillary zone electrophoresis (CZE)-MS/MS for high-resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS-PAGE gel and follow-up cleanup as well as CZE-MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high-resolution separation and characterization of histone proteoforms. SDS-PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high-resolution separations of SDS-PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems.