ELECTRON TRANSFER DISSOCIATION OF GLYCANS AND GLYCOCONJUGATES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 尽管电子捕获已经证明了它对蛋白质、多肽和碳水化合物的有效性，但它的应用受到了这样一个事实的限制，即它的使用几乎完全限于傅立叶变换离子回旋共振质谱仪。为了使基于电子的方法在FTMS仪器以外的仪器上实用，Hunt等人。介绍了一种新的解离方法，它依赖于负离子化学电离源中产生的负离子(通常是荧蒽阴离子)的电子转移，然后在气相中与由ESI产生并捕获在细胞中的多电荷正离子接触。鉴于我们在多糖的ECD和EDD方面取得的良好结果，我们开展了一项新的项目，探索ETD在分析多糖和糖偶联物方面的应用。由于我们在资源中还没有配备用于ETD的仪器，我们通过利用Bruker Daltonics，Inc.的报价开始了我们的研究，该公司在马萨诸塞州比勒里卡的总部提供了一台配备适当的四极离子陷阱仪器。我们最初的实验产生了具有丰富模式的光谱，其中包括糖苷和跨环裂解。因此，它们与我们已经获得的多糖的ECD谱有很大的相似之处，但也显示了额外的片段，因此数据是互补的。2009年4月，布鲁克在我们的实验室安装了他们的一台新的离子陷阱仪器(Amazon ETD)，这样我们就可以继续进行这些研究，还可以生成糖链的参考串联质谱图，我们将提供给公共数据库。对于天然化合物和天然、还原和O-18标记的类似物的全甲基化衍生物，用CID和ETD单独和组合获得了一组牛奶多糖的质谱图。这些都得到了详尽的解释，最终的手稿发表在《美国质谱学学会杂志》上。进一步的分析包括N-连接的多糖和糖肽。此外，正在评估用于ETD的化学试剂和化学电离气体的变化，以优化用于多糖的方法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Although ECD has proven its usefulness for proteins, peptides and carbohydrates, its applications are limited by the fact that its use is almost completely restricted to Fourier transform ion cyclotron resonance mass spectrometers. To make electron-based methods practical on instruments other than FTMS instruments, Hunt et al. introduced a new method for dissociation that relies on electron transfer by negative species (usually fluoranthene anions) that are produced in a negative-ion chemical ionization source and then brought into contact, in the gas phase, with multiply-charged positive ions that have been generated by ESI and trapped in a cell. Because of the excellent results that we had obtained by ECD and EDD of glycans, we undertook a new project to explore the utility of ETD for analysis of glycans and glycoconjugates. Since we did not yet have an instrument equipped for ETD in the Resource, we began our studies by taking advantage of an offer from Bruker Daltonics, Inc., who had an appropriately equipped quadrupole ion trap instrument available at their headquarters in nearby Billerica, MA. Our initial experiments produced spectra that have rich patterns that include both glycosidic and cross-ring cleavages. They therefore bear strong similarities to the ECD spectra of glycans that we have already obtained, but also exhibit additional fragments, and thus the data is complementary. In April 2009, Bruker installed one of their new ion trap instruments (amaZon ETD) in our laboratory, so that we may continue these studies and also generate reference tandem mass spectra for glycans that we will make available for inclusion in public databases. The mass spectra obtained by CID and ETD, alone and in combination, of a set of milk glycans have been obtained, for the native compounds, and for the permethylated derivatives of the native, reduced and O-18 labelled analogs. These were exhaustively interpreted and the resulting manuscript was published in the Journal of the American Society for Mass Spectrometry. Further analyses have included N-linked glycans and glycopeptides. In addition, variation of the chemical reagent for ETD and the chemical ionization gas is being evaluated to optimize the approach for glycans.