Extraction of Histones from Clinical Specimens for Epigenetic Profiling by Mass Spectrometry

Extensive experimental evidence supports the role of epigenetic mechanisms in cancer onset, progression, and recurrence. Among these, histone variants and their post-translational modifications (PTMs) regulate chromatin organization and dynamics, and genome accessibility, thereby influencing key DNA-based processes, such as transcription, replication, and DNA damage repair through the so-called histone code. Dysregulated histone PTM patterns are frequently observed in cancers, with some markers that have been shown to serve as diagnostic and prognostic biomarkers. Thus, novel methodologies enabling unbiased and comprehensive histone PTM profiling in clinical samples are highly valuable for characterizing the epigenetic landscapes of tumors. Here, we present a protocol for the efficient extraction of histones from clinical samples, including snap frozen, optimal-cutting temperature (OCT) frozen, and formalin-fixed paraffin-embedded (FFPE) biopsies. This approach yields histone proteins of sufficient quantity and quality for the subsequent bottom-up analysis by high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). This workflow enables the robust detection and quantification of key histone modifications, particularly lysine acetylations and methylations, across primary tumor samples, providing an additional molecular layer for cancer characterization.