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

ABSTRACT: Top-down mass spectrometry (TDMS) is a powerful tool to reveal the mechanism of epigenetic regulation by histones. However, the high similarity of histone variants and their highly dynamic post-translational modifications (PTMs) make them as on-going challenge analytes for separation with traditional chromatographic-based methods. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which enables high-resolution protein separation based on molecular size, not only split histone proteins in different channels, but also isolate the large intact histone proteins from the truncated ones. Here, for the first time, we employed SDS-PAGE integrating with the second-dimensional separation of capillary zone electrophoresis (CZE) to distinguish intact histone proteoforms containing different PTMs. Due to the unique physicochemical property of histone proteins regarding small proteins carrying highly positive charges, we systematically evaluated every step in the strategy, achieving high histone protein recovery with trichloroacetic acid precipitation, better separation with basic sample buffer as well as confident identification with low collision energy for histone proteins. The optimized workflow provides the distinguish of intact histone variants that differ by few amino acids near both the N- and C-termini as well as the identification of combinatorial PTMs and the crosstalk between PTMs.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Bos Taurus (bovine)

SUBMITTER: Liangliang Sun  

LAB HEAD: Liangliang Sun

PROVIDER: PXD048061 | Pride | 2024-10-15

REPOSITORIES: Pride