Background: 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. 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. Results: To 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. Conclusions: These 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.

Spatial epi-proteomics enabled by histone post-translational modification analysis from low-abundance clinical samples / R. Noberini, E.O. Savoia, S. Brandini, F. Greco, F. Marra, G. Bertalot, G. Pruneri, L.A. Mcdonnell, T. Bonaldi. - In: CLINICAL EPIGENETICS. - ISSN 1868-7083. - 13:1(2021 Dec), pp. 145.1-145.16. [10.1186/s13148-021-01120-7]

Spatial epi-proteomics enabled by histone post-translational modification analysis from low-abundance clinical samples

F. Marra
Membro del Collaboration Group
;
G. Pruneri
Membro del Collaboration Group
;
T. Bonaldi
Ultimo
Supervision
2021

Abstract

Background: 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. 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. Results: To 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. Conclusions: These 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.
Epigenetics; Histone post-translational modifications; Laser microdissection; MALDI-MSI; Mass spectrometry; Proteomics;
Settore BIO/13 - Biologia Applicata
Settore BIO/11 - Biologia Molecolare
Settore BIO/10 - Biochimica
dic-2021
28-lug-2021
Article (author)
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/910158
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