biostudies-literatureLopes MCrohn's and Colitis Foundation of AmericaNational Institute of Allergy and Infectious DiseasesEunice Kennedy Shriver National Institute of Child Health and Human DevelopmentNICHD NIH HHSNIAID NIH HHSNHLBI NIH HHSNational Cancer InstituteNCI NIH HHS5405-5420https://www.ebi.ac.uk/biostudies/studies/S-EPMC11932154biostudies-literatureUnknown23(12)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 tJournal of proteome researchOptimized and Robust Workflow for Quantifying the Canonical Histone Ubiquitination Marks H2AK119ub and H2BK120ub by LC-MS/MS.PMC11932154T32CA009140R01 HL177113R01 HD106051R01HD106051T32 CA009140P01 CA196539P01CA196539RFA 598467R01AI118891R01 AI118891Lopes MLund PJGarcia BAfalseOptimized 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 t2024-01-01T00:00:00Z2024 Dec2026-06-01T14:44:19.119Z2025-07-05T03:05:26.838ZS-EPMC119321543955665910.1021/acs.jproteome.4c00519