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.
Epigenetic regulation of chromatin is dependent on both the histone protein isoforms and state of their post-translational modifications. The assignment of all post-translational 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.
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158-2517, USA.
Shannon M
SM
Eliuk
David
D
Maltby
Barbara
B
Panning
Alma L
AL
Burlingame
D000595:Amino Acid Sequence; D000818:Animals; D005591:Chemical Fractionation / Q000379:methods*; D002853:Chromatography, Liquid; D004583:Electrons; D053595:Embryonic Stem Cells / Q000378:metabolism*; D006657:Histones / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009862:Online Systems; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational
D006657:Histones; D010455:Peptides; D020033:Protein Isoforms