Structural Analysis of 14-3-3-ζ-Derived Phosphopeptides Using Electron Capture Dissociation Mass Spectrometry, Traveling Wave Ion Mobility Spectrometry, and Molecular Modeling.
Structural Analysis of 14-3-3-ζ-Derived Phosphopeptides Using Electron Capture Dissociation Mass Spectrometry, Traveling Wave Ion Mobility Spectrometry, and Molecular Modeling.
复制标题
使用电子捕获解离质谱、行波离子淌度光谱和分子建模对 14-3-3-γ-衍生磷酸肽进行结构分析。
DOI：
10.1021/acs.jpcb.9b08506
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Cooper,HelenJ
中科院分区：
文献类型：
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作者：
Simmonds,AnnaL;Lopez-Clavijo,AndreaF;Winn,PeterJ;Russell,DavidH;Styles,IainB;Cooper,HelenJ
以前，我们已经证明了盐桥的电子捕获解离质谱行为的合成模型磷酸肽的效果，并应用离子迁移谱/分子建模方法，合理化的肽离子结构方面的调查结果。在这里，我们开发和应用的方法，以生物衍生的磷酸肽。具体而言，我们已经研究了衍生自Akt 1底物14-3-3-Arg的15聚体磷酸肽VVGARRSWRVVSSI（s表示磷酸化Ser）的变体，其含有磷酸化基序RRSWR。通过在磷酸化基序内连续的亮氨酸至亮氨酸取代产生变体。八种磷酸肽变体的ECD片段化模式显示出更大的序列覆盖度，具有连续的R → L取代。具有两个或更多个碱性残基的肽具有没有序列覆盖的区域，而对于具有一个或没有碱性残基的肽观察到全序列覆盖。对于三种肽变体，在磷酸丝氨酸和碱性残基之间观察到低丰度片段，这可能是由于存在多个构象异构体，这些残基之间存在或不存在非共价相互作用。对于解离行为表明存在分子内非共价相互作用的五种变体，我们采用离子迁移谱和分子建模来探测这些相互作用的性质。我们的工作流程使我们能够提出候选结构，其非共价相互作用与所有建模肽的ECD数据一致。此外，还提供了为这项工作创建并验证的AMBER参数集，并可在线获得（http：//www.biosciences-labs.bham.ac.uk/cooper/cubets.php）。
Previously, we have demonstrated the effect of salt bridges on the electron capture dissociation mass spectrometry behavior of synthetic model phosphopeptides and applied an ion mobility spectrometry/molecular modeling approach to rationalize the findings in terms of peptide ion structure. Here, we develop and apply the approach to a biologically derived phosphopeptide. Specifically, we have investigated variants of a 15-mer phosphopeptide VVGARRSsWRVVSSI (s denotes phosphorylated Ser) derived from Akt1 substrate 14-3-3-ζ, which contains the phosphorylation motif RRSsWR. Variants were generated by successive arginine-to-leucine substitutions within the phosphorylation motif. ECD fragmentation patterns for the eight phosphopeptide variants show greater sequence coverage with successive R → L substitutions. Peptides with two or more basic residues had regions with no sequence coverage, while full sequence coverage was observed for peptides with one or no basic residues. For three of the peptide variants, low-abundance fragments were observed between the phosphoserine and a basic residue, possibly due to the presence of multiple conformers with and without noncovalent interactions between these residues. For the five variants whose dissociation behavior suggested the presence of intramolecular noncovalent interactions, we employed ion mobility spectrometry and molecular modeling to probe the nature of these interactions. Our workflow allowed us to propose candidate structures whose noncovalent interactions were consistent with the ECD data for all of the peptides modeled. Additionally, the AMBER parameter sets created for and validated by this work are presented and made available online (http://www.biosciences-labs.bham.ac.uk/cooper/datasets.php).