Discrimination of Isomers of Released N- and O-Glycans Using Diagnostic Product Ions in Negative Ion PGC-LC-ESI-MS/MS
Discrimination of Isomers of Released N- and O-Glycans Using Diagnostic Product Ions in Negative Ion PGC-LC-ESI-MS/MS
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DOI：
10.1007/s13361-018-1932-z
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发表时间：
2018-06-01
影响因子：
3.2
通讯作者：
Packer, Nicolle H.
中科院分区：
文献类型：
--
作者：
Ashwood, Christopher;Lin, Chi-Hung;Packer, Nicolle H.
分析细胞蛋白质糖基化是具有挑战性的，因为存在高度相似的聚糖结构，在细胞生理学中发挥不同的作用。由于单个糖苷键的端基异构性和确切的键合类型会影响聚糖功能，因此需要改进的自动化方法来确认详细的结构特征并区分结构相似的异构体，从而克服糖组学实验产生的数据分析中的重大瓶颈。我们使用多孔石墨化碳-LC-ESI-MS/MS分离和检测释放的N-和O-聚糖异构体从哺乳动物模型糖蛋白使用负模式共振激活CID-MS/MS。通过询问相似的片段光谱，从密切相关的聚糖异构体，不同的只是在臂位置和唾液酸键，产品碎片离子，这些功能之间的歧视被发现。使用Skyline软件，验证了至少两种高特异性的诊断碎片离子，用于自动区分N-聚糖结构中的唾液酸化和臂位置以及O-聚糖结构中的唾液酸化，补充现有的结构诊断离子。当分析来自细胞裂解物的复杂聚糖混合物时，这些诊断离子显示出对于使用线性和3D离子阱质谱仪的异构体辨别是有用的。Skyline被认为是自动评估聚糖异构体歧视的有用工具。这种独立于平台的工作流程可以扩展到自动化其他具有挑战性的聚糖异构体的表征和定量。
Profiling cellular protein glycosylation is challenging due to the presence of highly similar glycan structures that play diverse roles in cellular physiology. As the anomericity and the exact linkage type of a single glycosidic bond can influence glycan function, there is a demand for improved and automated methods to confirm detailed structural features and to discriminate between structurally similar isomers, overcoming a significant bottleneck in the analysis of data generated by glycomics experiments. We used porous graphitized carbon-LC-ESI-MS/MS to separate and detect released N- and O-glycan isomers from mammalian model glycoproteins using negative mode resonance activation CID-MS/MS. By interrogating similar fragment spectra from closely related glycan isomers that differ only in arm position and sialyl linkage, product fragment ions for discrimination between these features were discovered. Using the Skyline software, at least two diagnostic fragment ions of high specificity were validated for automated discrimination of sialylation and arm position in N-glycan structures, and sialylation in O-glycan structures, complementing existing structural diagnostic ions. These diagnostic ions were shown to be useful for isomer discrimination using both linear and 3D ion trap mass spectrometers when analyzing complex glycan mixtures from cell lysates. Skyline was found to serve as a useful tool for automated assessment of glycan isomer discrimination. This platform-independent workflow can potentially be extended to automate the characterization and quantitation of other challenging glycan isomers.