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Signature Ions Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) for Specific and Confident Glycation Site Mapping in Therapeutic Proteins.
Journal of the American Society for Mass Spectrometry ( IF 3.2 ) Pub Date : 2020-02-20 , DOI: 10.1021/jasms.9b00101
Lei Wang 1 , Charles Nwosu 1 , Yunfan Gao 1 , Mei May Zhu 1
Affiliation  

Higher-energy collisional dissociation (HCD) is a well-established fragmentation technique in liquid chromatography tandem mass spectrometry (LC-MS/MS) and is used to study protein post translational modifications (PTMs) during peptide mapping. However, labile PTMs like glycosylation, glycation, sulfonylation, or phosphorylation tend to fragment earlier than peptide backbones under HCD. This leads to complicated MS/MS spectra, compromising data quality and downstream data interpretation. Electron-transfer/higher-energy collision dissociation (EThcD) has been used to analyze PTMs, but important components might be missed because of the increased duty cycle. To address this issue, modification-specific fragment ions formed in HCD experiments could be utilized to trigger EThcD analysis only for modified peptides. The trigger for EThcD was established by applying HCD with a high normalized collision energy, generating multiple informative Amadori derived lysine signature ions from a glycated peptide. These signature ions were then applied to trigger targeted EThcD for lysine glycation identification. This improved approach can further expand the characterization efforts of highly labile PTMs in therapeutic proteins.

中文翻译:

签名离子触发的电子转移/高能碰撞解离(EThcD),用于治疗性蛋白质的特异性和可信糖基化位点定位。

高能碰撞解离(HCD)是液相色谱串联质谱(LC-MS / MS)中一种行之有效的片段化技术,用于研究肽图绘制过程中的蛋白质翻译后修饰(PTM)。但是,在HCD下,不稳定的PTM(如糖基化,糖基化,磺酰化或磷酸化)倾向于比肽主链更早地断裂。这导致了复杂的MS / MS谱图,损害了数据质量和下游数据解释。电子转移/高能碰撞解离(EThcD)已用于分析PTM,但是由于占空比增加,可​​能会错过重要的组件。为了解决这个问题,在HCD实验中形成的修饰特异性片段离子可用于仅对修饰肽触发EThcD分析。EThcD的触发因素是通过以高归一化碰撞能量应用HCD并从糖基化肽中产生多个Amadori衍生的赖氨酸特征离子而建立的。然后将这些特征离子应用于触发靶向的EThcD,以进行赖氨酸糖基化鉴定。这种改进的方法可以进一步扩大治疗蛋白中高度不稳定的PTM的表征工作。
更新日期:2020-02-20
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