Stable-isotope-labeled Histone Peptide Library for Histone Post-translational Modification and Variant Quantification by Mass Spectrometry
Stable-isotope-labeled Histone Peptide Library for Histone Post-translational Modification and Variant Quantification by Mass Spectrometry
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DOI：
10.1074/mcp.o113.036459
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发表时间：
2014-09-01
影响因子：
7
通讯作者：
Garcia, Benjamin A.
中科院分区：
文献类型：
--
作者：
Lin, Shu;Wein, Samuel;Garcia, Benjamin A.
为了通过质谱法进行准确的组蛋白变体和翻译后修饰 (PTM) 定量，我们使用 Protein-Aqua (TM) 技术提供了一个包含 93 种合成肽的库。该文库包含代表组蛋白 H3 肽不同修饰形式的 55 种肽、代表 H4 肽的 23 种肽、代表典型 H2A 肽的 5 种肽、代表 H2A.Z 肽的 8 种肽以及 MacroH2A 和 H2A.X 的肽。这些肽的 PTM 包括赖氨酸单甲基化 (me1)、二甲基化 (me2) 和三甲基化 (me3)；赖氨酸乙酰化；精氨酸me1；丝氨酸/苏氨酸磷酸化；和N-末端乙酰化。该文库用丙酸酐进行化学衍生化，丙酸酐是一种广泛采用的组蛋白肽定量方案。随后，使用质谱提取离子色谱图量化检测效率。该文库具有广泛的检测效率，最低和最高效率的肽之间存在超过 1700 倍的差异。在本文中，我们描述了不同修饰对肽检测效率的影响，并提供了纠正 93 种组蛋白肽之间的检测偏差的资源。简而言之，检测效率与分子量、疏水性、碱度或修饰类型之间没有相关性。相同类型的修饰可能对检测效率产生非常不同的影响，具体取决于它们在肽中的位置。我们还观察到修饰之间的拮抗作用。在小鼠滋养层干细胞的研究中，我们利用肽库的检测效率来校正组蛋白 PTM/变体定量。对于大多数检查的组蛋白肽，校正后的数据并没有改变生物学结论，但确实改变了这些肽的相对丰度。对于低丰度组蛋白 H2A 变体 MacroH2A，校正后的数据得出的结论与未校正的数据不同。这里提出的肽库和检测效率可以作为促进表观遗传学和蛋白质组学领域研究的资源。
To facilitate accurate histone variant and post-translational modification (PTM) quantification via mass spectrometry, we present a library of 93 synthetic peptides using Protein-Aqua (TM) technology. The library contains 55 peptides representing different modified forms from histone H3 peptides, 23 peptides representing H4 peptides, 5 peptides representing canonical H2A peptides, 8 peptides representing H2A.Z peptides, and peptides for both macroH2A and H2A.X. The PTMs on these peptides include lysine mono-(me1), di- (me2), and tri-methylation (me3); lysine acetylation; arginine me1; serine/threonine phosphorylation; and N-terminal acetylation. The library was subjected to chemical derivatization with propionic anhydride, a widely employed protocol for histone peptide quantification. Subsequently, the detection efficiencies were quantified using mass spectrometry extracted ion chromatograms. The library yields a wide spectrum of detection efficiencies, with more than 1700-fold difference between the peptides with the lowest and highest efficiencies. In this paper, we describe the impact of different modifications on peptide detection efficiencies and provide a resource to correct for detection biases among the 93 histone peptides. In brief, there is no correlation between detection efficiency and molecular weight, hydrophobicity, basicity, or modification type. The same types of modifications may have very different effects on detection efficiencies depending on their positions within a peptide. We also observed antagonistic effects between modifications. In a study of mouse trophoblast stem cells, we utilized the detection efficiencies of the peptide library to correct for histone PTM/variant quantification. For most histone peptides examined, the corrected data did not change the biological conclusions but did alter the relative abundance of these peptides. For a low-abundant histone H2A variant, macroH2A, the corrected data led to a different conclusion than the uncorrected data. The peptide library and detection efficiencies presented here may serve as a resource to facilitate studies in the epigenetics and proteomics fields.