Publication Date

6-30-2022

Journal

Journal of Proteomics

DOI

0.1016/j.jprot.2022.104596

PMID

35489683

PMCID

PMC10165948

PubMedCentral® Posted Date

6-30-2023

PubMedCentral® Full Text Version

Author MSS

Published Open-Access

yes

Keywords

Animals, Chromatin, Epigenesis, Genetic, Histones, Mice, Protein Array Analysis, Protein Processing, Post-Translational, Epigenetics, RPPA, High-throughput, Post-translational modifications, Pluripotent stem cells, Breast cancer

Abstract

Epigenetic variation plays a significant role in normal development and human diseases including cancer, in part through post-translational modifications (PTMs) of histones. Identification and profiling of changes in histone PTMs, and in proteins regulating PTMs, are crucial to understanding diseases, and for discovery of epigenetic therapeutic agents. In this study, we have adapted and validated an antibody-based reverse phase protein array (RPPA) platform for profiling 20 histone PTMs and expression of 40 proteins that modify histones and other epigenomic regulators. The specificity of the RPPA assay for histone PTMs was validated with synthetic peptides corresponding to histone PTMs and by detection of histone PTM changes in response to inhibitors of histone modifier proteins in cell cultures. The useful application of the RPPA platform was demonstrated with two models: induction of pluripotent stem cells and a mouse mammary tumor progression model. Described here is a robust platform that includes a rapid microscale method for histone isolation and partially automated workflows for analysis of histone PTMs and histone modifiers that can be performed in a high-throughput manner with hundreds of samples. This RPPA platform has potential for translational applications through the discovery and validation of epigenetic states as therapeutic targets and biomarkers. SIGNIFICANCE: Our study has established an antibody-based reverse phase protein array platform for global profiling of a wide range of post-translational modifications of histones and histone modifier proteins. The high-throughput platform provides comprehensive analyses of epigenetics for biological research and disease studies and may serve as screening assay for diagnostic purpose or therapy development.

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