Optimized\nand Robust Workflow for Quantifying the\nCanonical Histone Ubiquitination Marks H2AK119ub and H2BK120ub by\nLC–MS/MS

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