Histone H4 N-Terminal Acetylation in Kasumi-1 Cells Treated with Depsipeptide Determined by Acetic Acid-Urea Polyacrylamide Gel Electrophoresis, Amino Acid Coded Mass Tagging, and Mass Spectrometry

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• 作者：Liwen Zhang ; Xiaodan Su ; Shujun Liu ; Amy R. Knapp ; Mark R. Parthun ; Guido Marcucci ; Michael A. Freitas
• 刊名：Journal of Proteome Research
• 出版年：2007
• 出版时间：January 2007
• 年：2007
• 卷：6
• 期：1
• 页码：81 - 88
• 全文大小：185K
• 年卷期：v.6,no.1(January 2007)
• ISSN：1535-3907

文摘

Disrupted patterns of acetylation and deacetylation of core histones play an important role in silencingtranscription of hematopoietic important genes in acute myeloid leukemia (AML). A thoroughinvestigation of these mechanisms and the response to pharmacologic modifiers will provide a betterunderstanding of the role of histone acetylation in leukemogenesis. We describe here an analyticalapproach that combines acid urea polyacrylamide gel electrophoresis (AU-PAGE), amino acid codedmass tagging (AACM), and mass spectrometry (MS) for the investigation of histone acetylation patterns.The combined approach was used to follow the dynamics of H4 acetylation in Kasumi-1 cells harboringthe fusion gene AML1/ETO shown to aberrantly recruit histone deacetylases (HDACs). The histones inKasumi-1 cells were labeled by growing the cells in media in which lysine was replaced with stableisotope-labeled lysine (Lys-D₄). Labeled and unlabeled cells were treated with depsipeptide and analyzedat different time points (0, 4, 8, 12, 24, and 48 h). The cells were mixed, the histone was extracted, andacetylated H4 isoforms were separated using AU-PAGE before in-gel trypsin digestion. The digestswere analyzed by MALDI-TOF MS. Peptides were identified by mass and isotope pattern. LC-MS/MSof Arg-C digests were also performed to verify the acetylation pattern for H4. The major pattern ofacetylation was determined as follows: initial acetylation at K16, followed by acetylation at K12, andfinally acetylation of either K8 and/or K5.