Optimized and Robust Workflow for Quantifying the Canonical Histone Ubiquitination Marks H2AK119ub and H2BK120ub by LC–MS/MS

The eukaryotic genome is packaged around histone proteins,
which
are subject to a myriad of post-translational modifications. By controlling
DNA accessibility and the recruitment of protein complexes that mediate
chromatin-related processes, these modifications constitute a key
mechanism of epigenetic regulation. Since mass spectrometry can easily
distinguish between these different modifications, it has become an
essential technique in deciphering the histone code. Although robust
LC–MS/MS methods are available to analyze modifications on
the histone N-terminal tails, routine methods for characterizing ubiquitin
marks on histone C-terminal regions, especially H2AK119ub, are less
robust. Here, we report the development of a simple workflow for the
detection and improved quantification of the canonical histone ubiquitination
marks H2AK119ub and H2BK120ub. The method entails a fully tryptic
digestion of acid-extracted histones, followed by derivatization with
heavy or light propionic anhydride. A pooled sample is then spiked
into oppositely labeled single samples as a reference channel for
relative quantification, and data is acquired using PRM-based nano-LC–MS/MS.
We validated our approach with synthetic peptides as well as treatments
known to modulate the levels of H2AK119ub and H2BK120ub. This new
method complements existing histone workflows, largely focused on
the lysine-rich N-terminal regions, by extending modification analysis
to other sequence contexts.