We hypothesized that intrinsic reactivities of Lys residues would be an additional factor to determine regioselectivity of histone acylation catalysts. To quantitatively compare intrinsic reactivities of histone's Lys residues in a nucleosome, we reacted a recombinant nucleosome with a Lys-reactive probe, sulfotetrafluorophenyl (STP)-alkyne 17 (Figure ), and acylated lysines by the probe were quantified by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). H2BK120 was acylated 1.9 times more than H2BK116 (Figure ): 23% and 12% yield for H2BK120 and 116, respectively. The significant difference in the intrinsic reactivity could rationalize why PEG-LANA-DSH catalyst selectively acylated H2BK120, while H2BK116 was more proximate to the catalytically active site. We also quantified reactivities of other Lys residues around the acidic patch, and found that H2BK108 was the most reactive Lys (97% yield, Figure ). To contrast, intrinsic reactivities of H2BK43 and K46 were exceptionally low (less than 2% yield, Figure ). It has been shown that reactivities of Lys residues in hydrophobic pockets are relatively high. However, because nucleosome does not have apparent hydrophobic pockets, local environments, such as hydrogen-bond formation between the Lys and water, nearby amino acids, or DNA may determine the intrinsic reactivity of each Lys residues in nucleosomes. (a) CER of DSH. For catalyst 1, CER was defined by the distance between acyl pyridinium carbon atom (colored in yellow) and the N-terminus nitrogen atom of the LANA moiety (colored in green). For catalysts 2 and 3, CER was defined by the distance between acyl pyridinium carbon atom (colored in yellow) and the α-carbon atom of Lys conjugated with DSH (colored in green). Radiuses of CER were predicted by MD simulations in explicit water. See Supporting Information (SI) for details. (b) The catalyst introduction position and target Lys residues. Ser13 and Arg7 of the X-ray crystal structure of the LANA-DSH (1)-nucleosome complex (PDB: 5GTC) were mutated to Lys by PyMOL version 2.5.4 mutagenesis command. The nitrogen atom of the N-terminus, the α-carbon atom of Arg7, or the α-carbon atom of Ser13 were marked with a red circle. (c) Distances (Å) between ɛ-nitrogen atoms of the indicated histone Lys residues (H2BK108, 116, and 120) and the nitrogen atom of N-terminal (N terminal), the α-carbon atom of Arg7 (R7), or the α-carbon atom of Ser13 (S13) in LANA, respectively, were measured using 100-ns MD trajectory of the LANA-nucleosome complex in explicit water (see methods for calculation and Figure