Top-Down Characterization of Heavily Modified Histones Using 193 nm Ultraviolet Photodissociation Mass Spectrometry

The
characterization of protein post-translational modifications
(PTMs) remains a significant challenge for traditional bottom-up proteomics
methods owing to the lability of PTMs and the difficulty of mapping
combinatorial patterns of PTMs based on analysis of small peptides.
These shortcomings have accelerated interest in top-down MS/MS methods
that focus on analysis of intact proteins. Simultaneous mapping of
all PTMs requires extensive sequence coverage to confidently localize
modifications. 193 nm ultraviolet photodissociation (UVPD) has been
shown to generate unparalleled sequence coverage for intact proteins
compared to traditional MS/MS methods. This study focuses on identification
and localization of PTMs of histones by UVPD, higher-energy collisional
dissociation (HCD), and the hybrid method electron-transfer/higher-energy
collision dissociation (EThcD) via a high throughput liquid chromatography–mass
spectrometry strategy. In total, over 500 proteoforms were characterized
among these three activation methods with 46% of the identifications
found in common by two or more activation methods. EThcD and UVPD
afforded more extensive characterization of proteoforms than HCD with
average gains in sequence coverage of 15% and C-scores
that doubled on average.