Zhang Kangling, Yau Peter M, Chandrasekhar Bhaskar, New Ron, Kondrat Richard, Imai Brian S, Bradbury Morton E
UCR Mass Spectrometry Facility, Department of Chemistry, University of California, Riverside, CA 92521, USA.
Proteomics. 2004 Jan;4(1):1-10. doi: 10.1002/pmic.200300503.
Histone acetylation and methylation play a critical role in transcription and gene regulation. Identification of sites of lysine acetylation and methylation in histones or other proteins by mass spectrometry (MS) is of increasing interest. In this paper, we report the use of MS to differentiate between peptides containing acetylated or tri-methylated lysines. High accuracy matrix-assisted laser desorption/ionization-time of flight MS gives better than five parts per million measurement accuracy, which is sufficient to verify acetylation and/or methylation. Electrospray ionization tandem mass spectrometry was used to assign modification sites and to differentiate acetylation from methylation. Typically, an immonium ion at m/z 98 corresponds to a mono-methylated lysine and an immonium ion at m/z 126 corresponds to an acetylated lysine. The neutral loss ion (MH(+)-59) is unique for a tri-methylated lysine. For a peptide with two or more modification sites of acetylation or tri-methylation or one site containing partial acetylation and tri-methylation, the a(2)-, b(2)-type ion is the characteristic index for an acetylated lysine whereas the b(2)-59 ion is indicative of a tri-methylated lysine in the N-terminus. The y-type ions and y-59 ions are characteristic of an acetylated lysine and a tri-methylated lysine at the C-terminus, respectively. We demonstrated that a lysine in a peptide modified by methylation or acetylation can be differentiated by MS using our method. Even if more then one lysine is present in a peptide and different modifications of this amino acid occur, they can be distinguished. This method was successful for the determination of the acetylation and methylation status of lysine 9 of histone H3 in chicken erythrocytes and human HeLa cell lines.
组蛋白乙酰化和甲基化在转录和基因调控中起着关键作用。通过质谱（MS）鉴定组蛋白或其他蛋白质中赖氨酸乙酰化和甲基化位点越来越受到关注。在本文中，我们报道了使用质谱来区分含有乙酰化或三甲基化赖氨酸的肽段。高精度基质辅助激光解吸/电离飞行时间质谱的测量精度优于百万分之五，这足以验证乙酰化和/或甲基化。电喷雾电离串联质谱用于确定修饰位点并区分乙酰化和甲基化。通常，质荷比为98的亚铵离子对应单甲基化赖氨酸，质荷比为126的亚铵离子对应乙酰化赖氨酸。中性丢失离子（MH(+)-59）是三甲基化赖氨酸所特有的。对于具有两个或更多乙酰化或三甲基化修饰位点或一个同时含有部分乙酰化和三甲基化位点的肽段，a(2)-、b(2)-型离子是乙酰化赖氨酸的特征指标，而b(2)-59离子则表明N端存在三甲基化赖氨酸。y型离子和y-59离子分别是C端乙酰化赖氨酸和三甲基化赖氨酸的特征离子。我们证明，使用我们的方法，质谱可以区分肽段中经甲基化或乙酰化修饰的赖氨酸。即使肽段中存在多个赖氨酸且该氨基酸发生了不同修饰，也能将它们区分开来。该方法成功用于测定鸡红细胞和人HeLa细胞系中组蛋白H3第9位赖氨酸的乙酰化和甲基化状态。
