Selective fragmentation of the N-glycan moiety and protein backbone of ribonuclease B on an Orbitrap Fusion Lumos Tribrid mass spectrometer
Selective fragmentation of the N-glycan moiety and protein backbone of ribonuclease B on an Orbitrap Fusion Lumos Tribrid mass spectrometer
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在 Orbitrap Fusion Lumos Tribrid 质谱仪上选择性断裂核糖核酸酶 B 的 N-聚糖部分和蛋白质骨架
DOI：
10.1002/rcm.8273
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发表时间：
2018-12-15
影响因子：
2
通讯作者：
Tian, Zhixin
中科院分区：
文献类型：
--
作者：
Li, Shasha;Zhou, Yue;Tian, Zhixin
基本原理方法完整糖蛋白的功能研究和应用需要蛋白质主链和聚糖部分的结构表征；前者已通过 CID 和 ExD 中蛋白质主链的选择性断裂得到成功证明；后者是否可以通过聚糖部分的选择性断裂来实现仍有待探索。对 RNase B 溶液进行电喷雾，并在 Orbitrap Fusion Lumos Tribrid 质谱仪上使用 CID、ETD、HCD、ETciD 和 EThcD 单独分离其丰度最高（电荷态 z = 16）的 GlcNAc(2)Man(n) (n = 5-9) 完整糖型并进行片段化；解离参数经过优化，可实现 N-聚糖部分和蛋白质主链的选择性断裂以及高序列覆盖率。分别使用蛋白质和 N-聚糖数据库搜索引擎 ProteinGoggle 和 GlySeeker 解释获得的光谱。结果 结论 通过探索所有五种方法的不同解离参数，在低碰撞能量下，HCD 和 EThcD 中都观察到 N-聚糖部分（蛋白质主链保持完整）的选择性断裂，但仅观察到少数匹配的产物离子；在高碰撞能量下观察到更全面的碎片（蛋白质主链丢失）。在所有五种解离方法中都观察到选择性蛋白质主链断裂。为了使用串联质谱法对完整 N-糖蛋白进行全面的结构表征，可以使用 HCD 和 EThcD 在低能和高能下互补地鉴定 N-聚糖部分的组成和拓扑结构；而氨基酸序列和糖位点可以使用 CID、ETD、HCD、ETciD 和 EThcD 及其最佳解离参数来识别。
Rationale Methods The functional study and application of an intact glycoprotein require the structural characterization of both the protein backbone and the glycan moiety; the former has been successfully demonstrated with selective fragmentation of the protein backbone in CID and ExD; whether the latter can be achieved with selective fragmentation of the glycan moiety remains to be explored. RNase B solution was electrosprayed and its intact glycoforms of GlcNAc(2)Man(n) (n = 5-9) with the highest abundance (charge state z = 16) were isolated individually and fragmented using CID, ETD, HCD, ETciD, and EThcD on the Orbitrap Fusion Lumos Tribrid mass spectrometer; the dissociation parameters were optimized for selective fragmentation of the N-glycan moiety and protein backbone as well as high sequence coverage. The obtained spectra were interpreted using the protein and N-glycan database search engines ProteinGoggle and GlySeeker, respectively. Results Conclusions With exploration of different dissociation parameters for all the five methods, selective fragmentation of the N-glycan moiety (the protein backbone staying intact) was observed in both HCD and EThcD at low collisional energies, but only a few matched product ions were observed; more comprehensive fragmentation was observed at high collisional energies (the protein backbone lost). Selective protein backbone fragmentation was observed in all the five dissociation methods. For comprehensive structural characterization of intact N-glycoproteins using tandem mass spectrometry, the composition and topology of the N-glycan moiety can be identified using HCD and EThcD complementarily at low and high energies; while the amino acid sequence and glycosite can be identified using CID, ETD, HCD, ETciD, and EThcD with their optimal dissociation parameters.