Fig. 1: Three strategies for histone analysis, including Nuc-MS for the direct interrogation of intact nucleosomes.

a, In the hypothetical nucleosome mixture (top), the Nuc-MS workflow (right) detects the colocalization of H3 and H4 methylation (blue and pink triangles) and determines that H2A ubiquitination (in orange) is present on a separate nucleosome. In contrast, traditional methods (left) use either protease-derived peptides or whole histones under denaturing conditions to detect histone PTMs, blurring the modification states of intact nucleosomes in a mixture. Nuc-MS detects proteoforms and their PTMs present in intact nucleosomes by employing top–down MS in native mode (right). b, Data from the three steps of Nuc-MS on an intact unmodified nucleosome. First, the mass of intact nucleosomes is measured (MS¹, O, observed average mass; T, theoretical mass; Δm, error). Second, a single nucleosome charge state (for example, 35+ ions highlighted in green) is isolated and activated by collisions with nitrogen to eject all intact histones and detect them simultaneously at isotopic resolution (MS², reporting monoisotopic masses). Third, each histone is isolated and further activated to create backbone fragmentation products that characterize the proteoforms, revealing PTMs or sequence events (MS³; blue flags indicate fragment ions matching uniquely to human histone H3.1, depicted as a graphical fragment map at the bottom; the green rectangle on the upper right highlights the intact precursor).