Garcia Benjamin A, Barber Cynthia M, Hake Sandra B, Ptak Celeste, Turner Fiona B, Busby Scott A, Shabanowitz Jeffrey, Moran Richard G, Allis C David, Hunt Donald F
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.
Biochemistry. 2005 Oct 4;44(39):13202-13. doi: 10.1021/bi050906n.
Phosphorylation of histone H3 is a hallmark event in mitosis and is associated with chromosome condensation. Here, we use a combination of immobilized metal affinity chromatography and tandem mass spectrometry to characterize post-translational modifications associated with phosphorylation on the N-terminal tails of histone H3 variants purified from mitotically arrested HeLa cells. Modifications observed in vivo on lysine residues adjacent to phosphorylated Ser and Thr provide support for the existence of the "methyl/phos", binary-switch hypothesis [Fischle, W., Wang, Y., and Allis, C. D. (2003) Nature 425, 475-479]. ELISA with antibodies selective for H3 at Ser10, Ser28, and Thr3 show reduced activity when adjacent Lys residues are modified. When used together, mass spectrometry and immunoassay methods provide a powerful approach for elucidation of the histone code and identification of histone post-translational modifications that occur during mitosis and other specific cellular events.
组蛋白H3的磷酸化是有丝分裂中的标志性事件，且与染色体凝聚相关。在此，我们运用固定化金属亲和色谱法与串联质谱法相结合的方法，来表征从有丝分裂停滞的HeLa细胞中纯化出的组蛋白H3变体N端尾部上与磷酸化相关的翻译后修饰。在体内观察到的与磷酸化的丝氨酸（Ser）和苏氨酸（Thr）相邻的赖氨酸残基上的修饰，为“甲基/磷酸”二元开关假说[菲施勒，W.，王，Y.，以及阿利斯，C. D.（2003年）《自然》425卷，475 - 479页]的存在提供了支持。当相邻的赖氨酸残基被修饰时，用对Ser10、Ser28和Thr3处的H3具有选择性的抗体进行酶联免疫吸附测定（ELISA），其活性会降低。质谱法和免疫测定法一起使用时，为阐明组蛋白编码以及鉴定有丝分裂和其他特定细胞事件期间发生的组蛋白翻译后修饰提供了一种强有力的方法。
