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Controlling Hydrogen Scrambling in Multiply Charged Protein Ions during Collisional Activation: Implications for Top-Down Hydrogen/Deuterium Exchange MS Utilizing Collisional Activation in the Gas Phase

机译:碰撞激活过程中控制多重电荷蛋白质离子中的氢加扰:利用气相中的碰撞激活作用对自上而下的氢/氘交换MS的影响

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摘要

Hydrogen exchange in solution combined with ion fragmentationnin the gas phase followed by MS detectionnemerged in recent years as a powerful tool to study highernorder protein structure and dynamics. However, a certainntype of ion chemistry in the gas phase, namely, internalnrearrangement of labile hydrogen atoms (the so-callednhydrogen scrambling), is often cited as a factor limitingnthe utility of this experimental technique. Although severalnstudies have been carried out to elucidate the roles playednby various factors in the occurrence and the extent ofnhydrogen scrambling, there is still no consensus as tonwhat experimental protocol should be followed to avoidnor minimize it. In this study we employ fragmentation ofnmass-selected subpopulations of protein ions to assessnthe extent of internal proton mobility prior to dissociation.nA unique advantage of tandem MS is that it not onlynprovides a means to map the deuterium content of proteinnions whose overall levels of isotope incorporation can benprecisely defined by controlling the mass selection window,nbut also correlates this spatial isotope distributionnwith such global characteristic as the protein ion chargenstate. Hydrogen scrambling does not occur when thencharge state of the precursor protein ions selected fornfragmentation is high. Fragment ions derived from bothnN- and C-terminal parts of the protein are equally unaffectednby scrambling. However, spatial distribution ofndeuterium atoms obtained by fragmenting low-chargedensitynprotein ions is consistent with a very high degreenof scrambling prior to the dissociation events. The extentnof hydrogen scrambling is also high when multistagenfragmentation is used to probe deuterium incorporationnlocally. Taken together, the experimental results providena coherent picture of intramolecular processes occurringnprior to the dissociation event and provide guidance fornthe design of experiments whose outcome is unaffectednby hydrogen scrambling.
机译:近年来,溶液中的氢交换与气相中的离子碎片结合,随后进行质谱检测成为了研究高级蛋白质结构和动力学的有力工具。然而,经常引用某种离子化学气相形式,即不稳定氢原子的内部重排(所谓的氢杂散)作为限制该实验技术实用性的因素。尽管已经进行了一些研究来阐明各种因素在氢加扰的发生和程度中所起的作用,但仍未达成共识,因为应遵循一些实验规程以避免或最小化它。在这项研究中,我们采用蛋白质选择的低质量子群的碎片化来评估解离前内部质子迁移的程度。n串联质谱的独特优势在于,它不仅提供了一种手段来绘制同位素总掺入水平的蛋白质中氘的含量可以通过控制质量选择窗口精确定义,但也可以使这种空间同位素分布与蛋白质离子电荷状态等全局特征相关。当选择用于片段化的前体蛋白离子的电荷状态较高时,不会发生氢加扰。来自蛋白质的N端和C端部分的碎片离子同样不受扰动的影响。然而,通过分解低电荷密度蛋白离子获得的氘原子的空间分布与解离事件之前的高度扰乱相一致。当使用多级碎裂法局部探测氘的掺入时,氢的加扰程度也很高。两者合计,实验结果提供了解离事件发生之前发生的分子内过程的连贯图片,并为实验设计提供了指导,而实验的结果不受氢扰乱的影响。

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  • 来源
    《Analytical Chemistry》 |2010年第3期|p.942-950|共9页
  • 作者单位

    Department of Chemistry, University of MassachusettssAmherst, 710 North Pleasant Street,Amherst, Massachusetts 01003;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
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  • 入库时间 2022-08-17 13:36:34

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