Enrichment and characterization of histones by two-dimensional hydroxyapatite/reversed-phase liquid chromatography-mass spectrometry.
Here we report a novel two-dimensional liquid chromatography-mass spectrometry (2D LC-MS) method that combines offline hydroxyapatite (HA) chromatography with online reversed-phase liquid chromatography-mass spectrometry (HA/RP LC-MS). The 2D LC-MS method was used to enrich and characterize histones and their posttranslational modifications. The 2D HA/RP LC-MS approach separates histones based on their relative binding affinity to DNA and relative hydrophobicity. HA/RP separations showed improvement in the number of histone isoforms detected as compared with one-dimensional RP LC-MS of acid-extracted histones. The improved histone fractionation resulted in better detection of lower abundant histone variants as well as their posttranslationally modified isoforms. Histones eluted from the HA/RP in the following order: H1, H2A/H2B heterodimers followed by H3/H4 heterotetramers, as predicted from their spatial organization in nucleosomes for binding affinity to DNA. Comparison between HA-purified and acid-extracted histones revealed similar histone profiles with the exception that the HA fractions showed a greater number of H1 isoforms. Two elution conditions were also examined: batch elution and salt gradient elution. Although both elution techniques were able to fractionate the histones sufficiently, the salt gradient approach has the most potential for purification of selected histone isoforms.
Su
Xiaodan
X
Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University College of Medicine, 460 W 12th Ave, Columbus, OH 43210, USA.
Jacob
Naduparambil K
NK
Amunugama
Ravindra
R
Hsu
Pang-Hung
PH
Fishel
Richard
R
Freitas
Michael A
MA
D056148:Chromatography, Reverse-Phase / Q000379:methods*; D017886:Durapatite / Q000737:chemistry*; D006657:Histones / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D020033:Protein Isoforms / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D006657:Histones; D020033:Protein Isoforms; D017886:Durapatite