title: Protein mass analysis of histones.
abstract: Posttranslational modification of chromatin-associated proteins, including histones and high-mobility-group (HMG) proteins, provides an important mechanism to control gene expression, genome integrity, and epigenetic inheritance. Protein mass analysis provides a rapid and unbiased approach to monitor multiple chemical modifications on individual molecules. This review describes methods for acid extraction of histones and HMG proteins, followed by separation by reverse-phase chromatography coupled to electrospray ionization mass spectrometry (LC/ESI-MS). Posttranslational modifications are detected by analysis of full-length protein masses. Confirmation of protein identity and modification state is obtained through enzymatic digestion and peptide sequencing by MS/MS. For differentially modified forms of each protein, the measured intensities are semiquantitative and allow determination of relative abundance and stoichiometry. The method simultaneously detects covalent modifications on multiple proteins and provides a facile assay for comparing chromatin modification states between different cell types and/or cellular responses.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Galasinski
authors.forename: Katheryn A
authors.initials: KA
authors.lastname: Resing
authors.forename: Natalie G
authors.initials: NG
authors.lastname: Ahn
mesh_terms: D000818:Animals; D002453:Cell Cycle; D002498:Centrifugation / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D006609:High Mobility Group Proteins / Q000737:chemistry / Q000378:metabolism; D006657:Histones / Q000737:chemistry* / Q000378:metabolism*; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: D006609:High Mobility Group Proteins; D006657:Histones