title: Analysis of human histone H4 by capillary electrophoresis in a pullulan-coated capillary, LC-ESI-MS and MALDI-TOF-MS.
abstract: The object of the present study was the analysis of the human histone H4 (a core histone) in order to evaluate the state of its acetylation. Capillary electrophoresis (CE) using a pullulan-coated capillary provides a rapid and efficient approach to the separation of monoacetylated, diacetylated and triacetylated H4 isoforms from human cells. By using a simple running buffer of 100 mM triethanolamine-phosphate solution at pH 2.5 and exploiting the effectiveness of pullulan-based coverage in preventing adsorptive phenomena, the separation of the differently acetylated isoforms was achieved in less than 15 min with high efficiency and reproducibility. The proposed method was for the first time applied in the analysis of histone H4 fractions obtained from cell lines treated with different histone deacetylase (HDAC) inhibitors, used as potential anticancer drugs. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) analysis demonstrated that the acetylation occurred in the histone H4 tail, whereas the CE separation allowed for a fast determination of the percentages of H4 acetylated isoforms in real samples; the results were in agreement with those obtained from liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS) analysis. Therefore, the proposed CE method is a useful complementary support to the hyphenated techniques for the rapid monitoring of the activity of HDAC inhibitors.
authors.affiliation: Department of Pharmaceutical Sciences, Alma Mater Studiorum-University of Bologna, Via Belmeloro 6, 40126, Bologna, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Olmo
authors.forename: Roberto
authors.initials: R
authors.lastname: Gotti
authors.forename: Marina
authors.initials: M
authors.lastname: Naldi
authors.forename: Vincenza
authors.initials: V
authors.lastname: Andrisano
authors.forename: Natalia
authors.initials: N
authors.lastname: Calonghi
authors.forename: Carola
authors.initials: C
authors.lastname: Parolin
authors.forename: Lanfranco
authors.initials: L
authors.lastname: Masotti
authors.forename: Vanni
authors.initials: V
authors.lastname: Cavrini
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D019075:Electrophoresis, Capillary; D005936:Glucans / Q000737:chemistry*; D006657:Histones / Q000032:analysis*; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013499:Surface Properties
chemical_list: D005936:Glucans; D006657:Histones; C009109:pullulan