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

(a) In the figure’s hypothetical nucleosome mixture (a, top), the Nuc-MS workflow (a, right side) detects the co-localization of H3 and H4 methylation (blue and pink triangles) and determines that the H2A ubiquitination (in orange) is present on a separate nucleosome. In contrast, traditional methods (a, left side) 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 (a, right side). (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 (e.g. 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 bottom; green rectangle in the upper right of the panel highlights the intact precursor).