Current proteomic approaches disassemble and digest nucleosome particles, blurring readouts of the ‘histone code’. To preserve nucleosome-level information, we developed Nuc-MS, which displays the landscape of histone variants and their post-translational modifications (PTMs) in a single mass spectrum. Combined with immunoprecipitation, Nuc-MS quantified nucleosome co-occupancy of histone H3.3 with variant H2A.Z (sixfold over bulk) and the co-occurrence of oncogenic H3.3K27M with euchromatic marks (for example, a >15-fold enrichment of dimethylated H3K79me2). Nuc-MS is highly concordant with chromatin immunoprecipitation-sequencing (ChIP-seq) and offers a new readout of nucleosome-level biology.
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This work was supported by the National Institute of General Medical Sciences (P41 GM108569) for the National Resource for Translational and Developmental Proteomics at Northwestern University and NIH grants S10OD025194 and RF1AG063903 (Kelleher laboratory) and R44GM116584, R44CA212733 and R44CA214076 (EpiCypher). L.F.S. is a Gilliam Fellow of the Howard Hughes Medical Institute. Research in this publication is also supported by Thermo Fisher Scientific and a fellowship associated with the Chemistry of Life Processes Predoctoral Training grant T32GM105538 at Northwestern University. A.P. is supported by the Transition to Independence grant K99CA234434-01. We also thank M. Senko, P. Compton, C. Koo, L. Szymczak and M. McAnnally for technical assistance and S. Judge and A. Rosenzweig for providing thoughtful suggestions to improve the manuscript.
N.L.K. serves as a consultant to Thermo Fisher Scientific. EpiCypher is a commercial developer and supplier of reagents, including the recombinant semi-synthetic modified nucleosomes (dNucs) used in this study. The other authors declare no competing interests.
Peer review information Arunima Singh was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–19, Tables 1–3 and Protocol.
Statistical source data and analysis for quantitative comparison of two equimolar synthetic Nucs presented in Supplementary Fig. 4b.
Statistical source data and analysis for quantitative comparison of H3.3 and HEK293T bulk Nucs presented in Supplementary Fig. 9.
Statistical source data and analysis for quantitative comparison of H3.3K27M fragments that localize to K79me2 in Supplementary Fig. 14.
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Schachner, L.F., Jooß, K., Morgan, M.A. et al. Decoding the protein composition of whole nucleosomes with Nuc-MS. Nat Methods 18, 303–308 (2021). https://doi.org/10.1038/s41592-020-01052-9