Shotgun annotation of histone modifications: A new approach for streamlined characterization of proteins by top down mass spectrometry
Shotgun annotation of histone modifications: A new approach for streamlined characterization of proteins by top down mass spectrometry
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DOI：
10.1021/ja039748i
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发表时间：
2004-03-24
影响因子：
15
通讯作者：
Kelleher, NL
中科院分区：
文献类型：
--
作者：
Pesavento, JJ;Kim, YB;Kelleher, NL
真核组蛋白是翻译后复杂性的典型例子，在许多不同的残基上具有不同的修饰（PTM），这些残基构成了“组蛋白密码”。为了帮助更有效地破解这一代码，我们展示了一种新的蛋白质表征策略，其中完整的PTM描述是通过数据库检索获得的，而不是通过高分辨率串联质谱（MS/MS）对信息丰富的数据进行手动解释。建立了近50 000个修饰组蛋白H4序列的数据库，并在四极傅里叶变换混合质谱仪中选择性地积累了来自组蛋白形式+112 Da（相对于未修饰质量）的电子捕获解离的91个碎片离子。检索列表顶部的正确形式表明Lys 20处二甲基化、N末端乙酰化和Lys 16处乙酰化（从三甲基化中分离，Δm= 0.036 Da）。统计评估揭示了质量准确性的关键作用，PTM“异构体”被检索为下一个最佳匹配。证明了鸟枪注释对具有多达六个PTM的H4形式的适用性，并可扩展到其他组蛋白（例如，H2 A，H2 B，H3）和其他蛋白质类预测。
Eukaryotic histones serve as prototypical examples of posttranslational complexity with diverse modifications (PTMs) on many different residues that comprise a “Histone Code”. To help crack this code more efficiently, we demonstrate a new strategy for protein characterization wherein complete PTM descriptions are obtained by database retrieval instead of manual interpretation of information-rich data from high-resolution tandem mass spectrometry (MS/MS). A database of nearly 50 000 modified histone H4 sequences was created and queried with 91 fragment ions from electron capture dissociation of a histone form +112 Da (versus unmodified mass) selectively accumulated in a quadrupole Fourier transform hybrid mass spectrometer. The correct form atop the retrieval list indicated dimethylation at Lys20, acetylation at the N terminus, and acetylation at Lys16 (resolved from trimethylation, Δm= 0.036 Da). A statistical evaluation reveals the critical role of mass accuracy and that PTM “isomers” are retrieved as next-best matches. The applicability of shotgun annotation to forms of H4 with up to six PTMs is demonstrated, with extensibility to other histones (e.g., H2A, H2B, H3) and other protein classes projected.