title: Cu2+-assisted two dimensional charge-mass double focusing gel electrophoresis and mass spectrometric analysis of histone variants.
abstract: Abundant isoforms and dynamic posttranslational modifications cause the separation and identification of histone variants to be experimentally challenging. To meet this need, we employ two-dimensional electrophoretic gel separation followed by mass spectrometric detection which takes advantage of the chelation of Cu(2+) with amino acid residues exposed on the surfaces of the histone proteins. Acid-extracted rat liver histones were first mixed with CuSO4 solution and then separated in one dimension with triton-acid-urea (TAU) gel electrophoresis and in a second dimension using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separations result from both the changes in charge and mass upon Cu(2+) chelation. Identities of each separated gel bands were obtained by using matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). It was found that the migration of H3 histone isoforms of rat liver is markedly affected by the use of Cu(2+) ions.
authors.affiliation: Key Laboratory of Pesticides and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, Hubei 430079, PR China.
authors.forename: Wenyang
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Pesticides and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, Hubei 430079, PR China.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Tang
authors.affiliation: Key Laboratory of Pesticides and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, Hubei 430079, PR China.
authors.forename: Mengjie
authors.initials: M
authors.lastname: Ding
authors.affiliation: Key Laboratory of Pesticides and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, Hubei 430079, PR China. Electronic address: hyzhong@mail.ccnu.edu.cn.
authors.forename: Hongying
authors.initials: H
authors.lastname: Zhong
mesh_terms: D000818:Animals; D003300:Copper / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006657:Histones / Q000032:analysis* / Q000302:isolation & purification; D008099:Liver / Q000737:chemistry*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D006657:Histones; D003300:Copper
keywords: Cu(2+) chelation; Gel electrophoresis; Histones; Mass spectrometry