Rapid identification of low level glycation sites in recombinant antibodies by isotopic labeling with 13C6-reducing sugars.
Rapid identification of low level glycation sites in recombinant antibodies by isotopic labeling with 13C6-reducing sugars.
复制标题
通过 13C6 还原糖同位素标记快速鉴定重组抗体中的低水平糖基化位点。
DOI：
10.1021/ac202995x
复制
发表时间：
2012
影响因子：
7.4
通讯作者：
V. Katta
中科院分区：
文献类型：
--
作者：
Jennifer Zhang;T. Zhang;Lihua Jiang;D. Hewitt;Yungfu Huang;Y. Kao;V. Katta
重组抗体由于在生产过程中接触还原糖而表现出低水平的糖化。由于糖化位点通常分布在整个抗体上，因此任何一个位点的水平都很低，并且很难在传统的肽图中检测到它们。通过与高浓度 (12)C(6)/(13)C(6) 还原糖的 1:1 混合物一起孵育，使模型抗体经历强制糖化，假设天然抗体中的相同位点将被糖化，但糖化程度较低。该方法通过给出具有相同强度且相差 6.018 Da 的两个分子离子的独特特征，简化了 LC/MS 分析中糖化胰蛋白酶肽洗脱的检测。内部开发的脚本自动处理大型数据文件以生成此类肽质量对的列表。 Orbitrap 的高质量准确度使我们能够通过与所有可能的糖化肽质量进行比较来明确分配序列。随后使用该序列表来验证它们在天然抗体消化物中是否存在。即使糖化水平低于 0.5%，该工作流程也能快速、可靠地识别位点特异性糖化。我们发现糖化位点分布在整个研究的抗体中。
Recombinant antibodies exhibit low levels of glycation from exposure to reducing sugars during production. As the glycation sites are typically distributed across the entire antibody, the levels at any one site are low and it becomes difficult to detect them in the conventional peptide maps. A model antibody was subjected to forced glycation by incubating with a high concentration of a 1:1 mixture of (12)C(6)/(13)C(6) reducing sugars with the assumption that the same sites in the native antibody will be glycated but to a lower extent. This approach simplified the detection of glycated tryptic peptide elution in the LC/MS analysis by giving a unique signature of two molecular ions with equal intensity and differing by 6.018 Da. An in-house developed script automatically processed large data files to generate a list of such peptide mass pairs. The high mass accuracy of the Orbitrap allowed us to assign the sequences unambiguously by comparison with all possible glycated peptide masses. This sequence list was subsequently used to verify their presence/absence in the digest of the native antibody. This work flow enabled rapid and confident identification of site-specific glycation even when levels are below 0.5%. We found the glycation sites to be distributed across the entire antibody studied.
影响因子：
4.4
作者：
Zhang, Qibin;Tang, Ning;Metz, Thomas O.
通讯作者：
Metz, Thomas O.
影响因子：
4.4
作者：
Zhang, Qibin;Tang, Ning;Metz, Thomas O.
通讯作者：
Metz, Thomas O.
影响因子：
7.4
作者：
Zhang, Qibin;Schepmoes, Athena A.;Brock, Jonathan W. C.;Wu, Si;Moore, Ronald J.;Purvine, Samuel O.;Baynes, John W.;Smith, Richard D.;Metz, Thomas O.
通讯作者：
Metz, Thomas O.