Separation and Characterization of Endogenous Nucleosomes by Native Capillary Zone Electrophoresis-Top-Down Mass Spectrometry.

ABSTRACT: We report a novel platform [native capillary zone electrophoresis-top-down mass spectrometry (nCZE-TDMS)] for the separation and characterization of whole nucleosomes, their histone subunits, and post-translational modifications (PTMs). As the repeating unit of chromatin, mononucleosomes (Nucs) are an ∼200 kDa complex of DNA and histone proteins involved in the regulation of key cellular processes central to human health and disease. Unraveling the covalent modification landscape of histones and their defined stoichiometries within Nucs helps to explain epigenetic regulatory mechanisms. In nCZE-TDMS, online Nuc separation is followed by a three-tier tandem MS approach that measures the intact mass of Nucs, ejects and detects the constituent histones, and fragments to sequence the histone. The new platform was optimized with synthetic Nucs to significantly reduce both sample requirements and cost compared to direct infusion. Limits of detection were in the low-attomole range, with linearity of over ∼3 orders of magnitude. The nCZE-TDMS platform was applied to endogenous Nucs from two cell lines distinguished by overexpression or knockout of histone methyltransferase NSD2/MMSET, where analysis of constituent histones revealed changes in histone abundances over the course of the CZE separation. We are confident the nCZE-TDMS platform will help advance nucleosome-level research in the fields of chromatin and epigenetics.

SUBMITTER: Jooß K 

PROVIDER: S-EPMC8040852 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature