Linker H1 Histone Direct Top-Down Proteoform Analysis

Linker H1 histones are highly susceptible to a variety of post-translational modifications (PTMs) that play a critical role in chromatin dynamics and gene expression. In the present work, trapped ion mobility spectrometry in tandem with ultraviolet photodissociation followed by ultrahigh resolution mass measurements, provided by a Fourier transform ion cyclotron mass spectrometer (TIMS-q-UVPD-FT-ICR MS/MS), enabled direct characterization of the linker H1 histone proteoforms. The proposed method takes advantage of the mobility and mass preseparation of core and linker histones proteoforms prior to UVPD fragmentation and consequent high mass accuracy detection in the FT-ICR MS of the fragment ions for efficient PTM assignment. The application to a bovine histone extraction resulted in the annotation of four H1 variants (H1.2, H1.3, H1.5, and H1.4V), together with their PTM distribution with high sequence coverages (up to 60%); in particular, the H1.4V variant sequence was identified via <i>de novo</i> sequencing. All reported H1 proteoforms and their PTMs (e.g., mono/dimethylation, acetylation, and phosphorylation) were confirmed with complementary top-down liquid chromatography coupled to tandem MS/MS using electron-activated dissociation (LC-q-EAD-ToF MS/MS) and bottom-up analysis. This direct approach shows great promise for global H1 proteoform analysis due minimal sample preparation and large number of proteoform PTM observed.