In this chapter, we describe the proteomic approach named “Native Chromatin Proteomics” (N-ChroP) that couples a modified Chromatin ImmunoPrecipitation (ChIP) protocol with the mass spectrometry (MS) analysis of immunoprecipitated proteins to study the combinatorial enrichment or exclusion of histone post-translational modifications (PTMs) at specific genomic regions, such as promoters or enhancers. We describe the protocol steps from the digestion of chromatin and nucleosome immunoprecipitation to histone digestion and peptide enrichment prior to MS analysis, up to the MS raw data analysis. We also discuss current challenges and offer suggestions based on the direct hands-on experience acquired during the method setup.

Native Chromatin Proteomics (N-ChroP) to Characterize Histone Post-translational Modification (PTM) Combinatorics at Distinct Genomic Regions / L. Nicosia, T. Bonaldi (METHODS IN MOLECULAR BIOLOGY). - In: Enhancers and Promoters / [a cura di] T. Borggrefe, B.D. Giaimo. - Prima edizione. - [s.l] : Humana Press Inc., 2021. - ISBN 978-1-0716-1596-6. - pp. 251-274 [10.1007/978-1-0716-1597-3_14]

Native Chromatin Proteomics (N-ChroP) to Characterize Histone Post-translational Modification (PTM) Combinatorics at Distinct Genomic Regions

L. Nicosia
Primo
Writing – Original Draft Preparation
;
T. Bonaldi
Ultimo
Writing – Review & Editing
2021

Abstract

In this chapter, we describe the proteomic approach named “Native Chromatin Proteomics” (N-ChroP) that couples a modified Chromatin ImmunoPrecipitation (ChIP) protocol with the mass spectrometry (MS) analysis of immunoprecipitated proteins to study the combinatorial enrichment or exclusion of histone post-translational modifications (PTMs) at specific genomic regions, such as promoters or enhancers. We describe the protocol steps from the digestion of chromatin and nucleosome immunoprecipitation to histone digestion and peptide enrichment prior to MS analysis, up to the MS raw data analysis. We also discuss current challenges and offer suggestions based on the direct hands-on experience acquired during the method setup.
Chromatin; Chromatin immunoprecipitation; Genomic regions; Histone post-translational modifications; Label-free quantification; Mass spectrometry; Proteomics; Chromatin; Chromatin Immunoprecipitation; Chromatography, Liquid; DNA-Binding Proteins; Data Analysis; Histones; Nucleosomes; Tandem Mass Spectrometry; Genomics; Protein Processing, Post-Translational; Proteomics
Settore BIO/11 - Biologia Molecolare
Settore BIO/13 - Biologia Applicata
Settore BIO/10 - Biochimica
2021
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/947949
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