High resolution electron transfer dissociation studies of unfractionated intact histones from murine embryonic stem cells using on-line capillary LC separation: Determination of abundant histone isoforms and post-translational modifications

Abstract

Epigenetic regulation of chromatin is dependent on both the histone protein isoforms and state of their post-trans-lational modifications. The assignment of all post-trans-lational modification sites for each individual intact protein isoform remains an experimental challenge. We present an on-line reversed phase LC tandem mass spectrometry approach for the separation of intact, unfractionated histones and a high resolution mass analyzer, the Orbitrap, with electron transfer dissociation capabilities to detect and record accurate mass values for the molecular and fragment ions observed. From a single LC-electron transfer dissociation run, this strategy permits the identification of the most abundant intact proteins, determination of the isoforms present, and the localization of post-translational modifications. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.