Isolation of Proteins on Nascent Chromatin and Characterization by Quantitative Mass Spectrometry.

Abstract

Replication-coupled chromatin assembly is a very dynamic process that involves not only the replication fork machinery but also chromatin-related factors such as histones, histone chaperones, histone-modifying enzymes, and chromatin remodelers which ensure not only that the genetic information is properly replicated but also that the epigenetic code is reestablished in the daughter cell. Of the histone modifications associated with chromatin assembly, acetylation is the most abundant. Determining how newly synthesized histones get acetylated and what factors affect this modification is vital to understanding how cells manage to properly duplicate the epigenome. Here we describe a combination of the iPOND, quantitative mass spectrometry, and SILAC methodologies to study the protein composition of newly assembled chromatin and the modification state of the associated histones.