PAT-H-MS coupled with laser microdissection to study histone post-translational modifications in selected cell populations from pathology samples

Giancarlo Pruneri,Roberta Noberini,Tiziana Bonaldi,Giuliana Pelicci,Cristina Richichi,Rémi Longuespée,Mark Kriegsmann

doi:10.1186/s13148-017-0369-8

Abstract

BackgroundAberrations in histone post-translational modifications (hPTMs) have been linked with various pathologies, including cancer, and could not only represent useful biomarkers but also suggest possible targetable epigenetic mechanisms. We have recently developed an approach, termed pathology tissue analysis of histones by mass spectrometry (PAT-H-MS), that allows performing a comprehensive and quantitative analysis of histone PTMs from formalin-fixed paraffin-embedded pathology samples. Despite its great potential, the application of this technique is limited by tissue heterogeneity.MethodsIn this study, we further implemented the PAT-H-MS approach by coupling it with techniques aimed at reducing sample heterogeneity and selecting specific portions or cell populations within the samples, such as manual macrodissection and laser microdissection (LMD).ResultsWhen applied to the analysis of a small set of breast cancer samples, LMD-PAT-H-MS allowed detecting more marked changes between luminal A-like and triple negative patients as compared with the classical approach. These changes included not only the already known H3 K27me3 and K9me3 marks, but also H3 K36me1, which was found increased in triple negative samples and validated on a larger cohort of patients, and could represent a potential novel marker distinguishing breast cancer subtypes.ConclusionsThese results show the feasibility of applying techniques to reduce sample heterogeneity, including laser microdissection, to the PAT-H-MS protocol, providing new tools in clinical epigenetics and opening new avenues for the comprehensive analysis of histone post-translational modifications in selected cell populations.

Highlights

Aberrations in histone post-translational modifications have been linked with various pathologies, including cancer, and could represent useful biomarkers and suggest possible targetable epigenetic mechanisms
We have reported for the first time a method that allows the MS-based analysis of histone post-translational modifications (hPTMs) from human pathology tissues, termed pathology tissue analysis of histones by mass spectrometry (PAT-H-MS) [8], which combines protocols used for global proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues [9] with a proteomic workflow optimized for hPTM analysis [10]
The yield is lower starting from stained sections, the SDS-PAGE run of the two samples shows very similar protein patterns and purity (Fig. 1a, bottom), and the amount obtained from hematoxylin and eosin (H&E)-stained sections is anyhow largely sufficient for MS-based hPTM analysis, which usually requires 4 μg of histones per run

Summary

Introduction

Aberrations in histone post-translational modifications (hPTMs) have been linked with various pathologies, including cancer, and could represent useful biomarkers and suggest possible targetable epigenetic mechanisms. Increasing evidence has linked aberrations in hPTMs with various pathologies, including cancer, suggesting. Noberini et al Clinical Epigenetics (2017) 9:69 could help uncover possible epigenetic mechanisms underlying different pathologies and provide novel epigenetic pathways targetable for therapy. The potential offered by the MS-based analysis of hPTMs in clinical cancer samples has been left largely unexploited. Most of the studies employing MS-based techniques, which can provide a comprehensive and quantitative view on hPTM patters, have focused on cell lines and animal tissue, while clinical samples are usually tested through antibody-based techniques

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