Improved Mass Spectrometry–Based Methods Reveal Abundant Propionylation and Tissue-Specific Histone Propionylation Profiles

Histone posttranslational modifications (PTMs) have crucial roles in a multitude of cellular processes, and their aberrant levels have been linked with numerous diseases, including cancer. Although histone PTM investigations have focused so far on methylations and acetylations, alternative long-chain acylations emerged as new dimension, as they are linked to cellular metabolic states and affect gene expression through mechanisms distinct from those regulated by acetylation. Mass spectrometry is the most powerful, comprehensive, and unbiased method to study histone PTMs. However, typical mass spectrometry–based protocols for histone PTM analysis do not allow the identification of naturally occurring propionylation and butyrylation. Here, we present improved state-of-the-art sample preparation and analysis protocols to quantitate these classes of modifications. After testing different derivatization methods coupled to protease digestion, we profiled common histone PTMs and histone acylations in seven mouse tissues and human normal and tumor breast clinical samples, obtaining a map of propionylations and butyrylations found in different tissue contexts. A quantitative histone PTM analysis also revealed a contribution of histone acylations in discriminating different tissues, also upon perturbation with antibiotics, and breast cancer samples from the normal counterpart. Our results show that profiling only classical modifications is limiting and highlight the importance of using sample preparation methods that allow the analysis of the widest possible spectrum of histone modifications, paving the way for deeper insights into their functional significance in cellular processes and disease states. [Display omitted] •Histone acylation has emerged as an important new dimension in genome regulation.•Propionylations and butyrylations cannot be analyzed using current digestion protocols.•We tested alternative digestion protocols to overcome this limitation.•Histone propionylation is a frequent and abundant modification.•Histone acylations discriminate different mouse and human tissues. Optimized histone in-gel derivatization coupled to protease digestion protocols for mass spectrometry analysis of histone posttranslational modifications enabled the definition of a catalog of propionylations and butyrylations found in vivo in different tissue contexts. A quantitative analysis also revealed a contribution of these modifications in discriminating different mouse tissues