Mass Spectrometry-based Profiling of Single-cell Histone Post-translational Modifications to Dissect Chromatin Heterogeneity

ABSTRACT: The emerging popularity of single-cell sequencing technologies has revealed unexpected heterogeneity of chromatin states and gene expression in seemingly homogeneous cell populations. Similarly, single-cell proteomics has proven capable of quantifying heterogeneity of proteomes amongst single cells, yet, due to their low stoichiometry, it remains limited in investigating the heterogeneity of protein post-translational modifications (PTMs). Here, we present a robust mass spectrometry-based method for the unbiased analysis of hPTMs at single-cell level, in order to investigate the complex relationship between the regulation of epigenetic marks and cell identity. We show that our method can identify both single and combinatorial hPTMs (68 peptidoforms in total), which includes nearly all of the most studied hPTMs. Our single-cell method achieves technical reproducibility comparable to traditional bulk histone experiments, and distinguishes between technical noise and biological cell-to-cell variation of hPTM abundances. As a proof of concept, we treated cells with sodium butyrate, a histone deacetylase inhibitor, and demonstrate that our method can i) distinguish between treated and non-treated cells , ii) identify sub-populations of cells that respond differently to treatment, and iii) reveal how hPTM regulation differs in the context of drug treatment using covariation analysis. Overall, these experiments establish the applicability of our method to investigate chromatin heterogeneity at the single-cell level, which has important implications for understanding complex disease states like cancer and aging. This innovative method provides new opportunities for analyzing the histone code by utilizing cellular heterogeneity to uncover regulatory principles.

INSTRUMENT(S): timsTOF Ultra

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Simone Sidoli  

PROVIDER: MSV000094801 | MassIVE | Sat May 18 19:18:00 BST 2024

SECONDARY ACCESSION(S): PXD052370

REPOSITORIES: MassIVE