Mass spectrometry-based profiling of single-cell histone post-translational modifications to dissect chromatin heterogeneity

Single-cell proteomics confidently quantifies cellular heterogeneity, however quantification of post-translational modifications, such as those deposited on histone proteins, remains elusive. Here, we develop a robust mass spectrometry-based method for the unbiased analysis of single-cell histone post-translational modifications (sc-hPTM). sc-hPTM identifies both single- and combinatorial histone post-translational modifications (67 peptidoforms in total), which includes nearly all frequently studied histone post-translational modifications with comparable reproducibility to traditional bulk experiments. As a proof of concept, we treat cells with sodium butyrate, a histone deacetylase inhibitor, and demonstrate that our method can i) distinguish between treated and untreated cells, ii) identify sub-populations of cells with heterogeneous response to the treatment, and iii) reveal differential co-regulation of histone post-translational modifications in the context of drug treatment. The sc-hPTM method enables comprehensive investigation of chromatin heterogeneity at single-cell resolution and provides a further understanding of the histone code. Chromatin structure is regulated by chemical modifications of histone proteins, but measuring these at single-cell resolution has been challenging. Here, the authors develop a mass spectrometry-based method to profile histone modifications in individual cells, revealing chromatin heterogeneity and differential co-regulation.