The use of a xylosylated plant glycoprotein as an internal standard accounting for N-linked glycan cleavage and sample preparation variability.
The use of a xylosylated plant glycoprotein as an internal standard accounting for N-linked glycan cleavage and sample preparation variability.
复制标题
使用木糖基化植物糖蛋白作为内标，解释 N-连接聚糖裂解和样品制备变异性。
DOI：
10.1002/rcm.6579
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Muddiman,DavidC
中科院分区：
文献类型：
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作者：
Walker,SHunter;Taylor,AmberD;Muddiman,DavidC
理论依据传统上，游离寡糖内标物用于解释质谱法聚糖相对定量实验中的变异性。然而，更合适的内标物是糖蛋白，它也可以控制酶裂解效率，从而允许更准确的定量实验。（天然和稳定同位素标记）用于衍生化和差异标记N-连接聚糖样品以进行相对定量，通过与Q-Exactive质谱仪在线连接的反相液相色谱芯片系统分析样品。研究了两种内标物，麦芽七糖（先前使用）和辣根过氧化物酶（HRP）（新），以证明在聚糖相对定量实验中使用糖蛋白作为内标物的有效性。因此，可以检测内标物木糖基化聚糖，而不干扰样品。此外，还表明可以通过监测HRP聚糖来检测切割效率的差异。在一个样品中，切割效率的变化是最小的，HRP聚糖执行以及maltoheptaose. CONCLUSIONS因为HRP聚糖执行以及maltoheptaose，但也能够纠正和解释切割的变异性，它是一个更通用的内标，并将用于所有后续的生物学研究。由于酶可能存在批间变异、生物基质差异和酶活性随时间变化，因此有必要在聚糖相对定量实验中考虑聚糖裂解变异性。版权所有© 2013约翰威利父子有限公司.
RATIONALETraditionally, free oligosaccharide internal standards are used to account for variability in glycan relative quantification experiments by mass spectrometry. However, a more suitable internal standard would be a glycoprotein, which could also control for enzymatic cleavage efficiency, allowing for more accurate quantitative experiments.METHODSHydrophobic, hydrazideN‐linked glycan reagents (both native and stable‐isotope labeled) are used to derivatize and differentially labelN‐linked glycan samples for relative quantification, and the samples are analyzed by a reversed‐phase liquid chromatography chip system coupled online to a Q‐Exactive mass spectrometer. The inclusion of two internal standards, maltoheptaose (previously used) and horseradish peroxidase (HRP) (novel), is studied to demonstrate the effectiveness of using a glycoprotein as an internal standard in glycan relative quantification experiments.RESULTSHRP is a glycoprotein containing a xylosylatedN‐linked glycan, which is unique from mammalianN‐linked glycans. Thus, the internal standard xylosylated glycan could be detected without interference to the sample. Additionally, it was shown that differences in cleavage efficiency can be detected by monitoring the HRP glycan. In a sample where cleavage efficiency variation is minimal, the HRP glycan performs as well as maltoheptaose.CONCLUSIONSBecause the HRP glycan performs as well as maltoheptaose but is also capable of correcting and accounting for cleavage variability, it is a more versatile internal standard and will be used in all subsequent biological studies. Because of the possible lot‐to‐lot variation of an enzyme, differences in biological matrix, and variable enzyme activity over time, it is a necessity to account for glycan cleavage variability in glycan relative quantification experiments. Copyright © 2013 John Wiley & Sons, Ltd.