Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments
Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments
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DOI：
10.1074/mcp.o115.053843
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发表时间：
2016-03-01
影响因子：
7
通讯作者：
Hunt, Donald F.
中科院分区：
文献类型：
--
作者：
Anderson, Lissa C.;Karch, Kelly R.;Hunt, Donald F.
组蛋白代表一类非常适合通过自上而下质谱法进行分析的蛋白质，这是由于其相对小的尺寸、其所表现出的高电子转移解离相容电荷状态以及获得关于组合翻译后修饰和变体的有价值信息的潜力。我们最近描述了质谱分析中用于获得完整蛋白质的高质量MS/MS谱的新方法（安德森，L. C.的方法，English，A. M.，王伟，Bai，白氏D. L.，Shabanowitz，J.，和Hunt，D. F.等人（2015）Int. J. Mass Spectrom. 377，617-624）。在这里，我们报告这些技术的扩展。在改进的Orbitrap Velos Pro/Elite（TM）中进行的连续离子/离子反应能够与数据依赖性和靶向HPLC-MS实验相结合，以获得来自丁酸盐处理的HeLa细胞的组蛋白的高分辨率MS/MS谱，所述改进的Orbitrap Velos Pro/Elite（TM）能够对C阱进行多个碎片离子填充。这些光谱用于鉴定几种独特的完整组蛋白蛋白质型，其序列覆盖率高达81%。我们还表明，平行离子停车在离子/离子质子转移反应过程中，可用于分离的重叠的m/z，不分离色谱，揭示以前难以辨别的信号的物种。最后，我们的特点是几个截断形式的H2 A和H2 B中发现的组蛋白组分分析，实现高达93%的序列覆盖率的电子转移解离MS/MS。后续的体外实验的结果表明，我们观察到的一些截断组蛋白H2 A proteoforms可以产生的组织蛋白酶L，一种酶也催化剪切组蛋白H3。
Histones represent a class of proteins ideally suited to analyses by top-down mass spectrometry due to their relatively small size, the high electron transfer dissociation-compatible charge states they exhibit, and the potential to gain valuable information concerning combinatorial post-translational modifications and variants. We recently described new methods in mass spectrometry for the acquisition of high-quality MS/MS spectra of intact proteins (Anderson, L. C., English, A. M., Wang, W., Bai, D. L., Shabanowitz, J., and Hunt, D. F. (2015) Int. J. Mass Spectrom. 377, 617-624). Here, we report an extension of these techniques. Sequential ion/ion reactions carried out in a modified Orbitrap Velos Pro/Elite(TM) capable of multiple fragment ion fills of the C-trap, in combination with data-dependent and targeted HPLC-MS experiments, were used to obtain high resolution MS/MS spectra of histones from butyrate-treated HeLa cells. These spectra were used to identify several unique intact histone proteoforms with up to 81% sequence coverage. We also demonstrate that parallel ion parking during ion/ion proton transfer reactions can be used to separate species of overlapping m/z that are not separated chromatographically, revealing previously indiscernible signals. Finally, we characterized several truncated forms of H2A and H2B found within the histone fractions analyzed, achieving up to 93% sequence coverage by electron transfer dissociation MS/MS. Results of follow-up in vitro experiments suggest that some of the truncated histone H2A proteoforms we observed can be generated by cathepsin L, an enzyme known to also catalyze clipping of histone H3.