Abstract
BackgroundIncreasing evidence linking epigenetic mechanisms and different diseases, including cancer, has prompted in the last 15 years the investigation of histone post-translational modifications (PTMs) in clinical samples. Methods allowing the isolation of histones from patient samples followed by the accurate and comprehensive quantification of their PTMs by mass spectrometry (MS) have been developed. However, the applicability of these methods is limited by the requirement for substantial amounts of material.ResultsTo address this issue, in this study we streamlined the protein extraction procedure from low-amount clinical samples and tested and implemented different in-gel digestion strategies, obtaining a protocol that allows the MS-based analysis of the most common histone PTMs from laser microdissected tissue areas containing as low as 1000 cells, an amount approximately 500 times lower than what is required by available methods. We then applied this protocol to breast cancer patient laser microdissected tissues in two proof-of-concept experiments, identifying differences in histone marks in heterogeneous regions selected by either morphological evaluation or MALDI MS imaging.ConclusionsThese results demonstrate that analyzing histone PTMs from very small tissue areas and detecting differences from adjacent tumor regions is technically feasible. Our method opens the way for spatial epi-proteomics, namely the investigation of epigenetic features in the context of tissue and tumor heterogeneity, which will be instrumental for the identification of novel epigenetic biomarkers and aberrant epigenetic mechanisms.
Highlights
Increasing evidence linking epigenetic mechanisms and different diseases, including cancer, has prompted in the last 15 years the investigation of histone post-translational modifications (PTMs) in clinical samples
Experimental procedures involving animals were performed in accordance with the Italian Laws (D.lgs. 26/2014), which enforces Dir. 2010/63/EU (‘‘Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes’’)
We chose to focus on an ingel, rather than an in-solution, protocol because SDSPAGE serves both to eliminate the detergents and other mass spectrometry (MS) contaminants (e.g., the optimal cutting temperature (OCT) compound) present in the histones enriched from clinical samples, and to separate the histones from the other proteins present in the extracts [14]
Summary
Increasing evidence linking epigenetic mechanisms and different diseases, including cancer, has prompted in the last 15 years the investigation of histone post-translational modifications (PTMs) in clinical samples. Many epigenetic mechanisms underlying cancer still need to be elucidated in order to identify novel targets, and sensitive biomarkers are needed to better guide patient diagnosis and their treatment with the most appropriate epigenetic drugs. In this scenario, an accurate and quantitative evaluation of histone PTM levels in clinical samples is becoming increasingly needed, as it can provide biomarkers useful for patient stratification, and suggest possible novel epigenetic mechanisms altered in cancer, which could be targeted for therapy
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