Identification of Chromatin Readers Using a Peptide Pull-Down and Mass Spectrometry Integrated Approach

Histone proteins regulate essential cellular processes by being decorated with a myriad of posttranslational modifications (PTMs). These processes are mostly led by the ability of these PTMs to recruit protein readers involved in gene transcription, DNA replication, DNA damage, chromatin remodeling, and other functions. Identifying histone readers is critical for the understanding of mechanisms leading to these functions and potentially predict targets for treatment in anomalous phenotypes. For this reason, histone reader identification has been performed for several years using strategies aiming to increase depth, resolution, and accuracy, e.g., ChIP-MS, proximity biotinylation, photo-crosslinking, and array-based technologies. In this chapter, we describe a protocol for identifying histone readers in a straightforward and unbiased manner: peptide pull-down combined with high-resolution mass spectrometry. Synthesized and immobilized modified histone peptides are incubated with nuclear extracts, and the PTMs' interactors are analyzed by mass spectrometry, which allows the identification of thousands of proteins with high confidence. We also describe the steps for a proteomic data analysis and present tools for a comprehensive data integration.