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Parallel Glyco-SPOT Synthesis of Glycopeptide Libraries.
Cell Chemical Biology ( IF 8.6 ) Pub Date : 2020-06-30 , DOI: 10.1016/j.chembiol.2020.06.007
Akul Y Mehta 1 , Ravi Kumar H Veeraiah 2 , Sucharita Dutta 1 , Christoffer K Goth 1 , Melinda S Hanes 1 , Chao Gao 1 , Kathrin Stavenhagen 1 , Robert Kardish 1 , Yasuyuki Matsumoto 1 , Jamie Heimburg-Molinaro 1 , Michael Boyce 3 , Nicola L B Pohl 2 , Richard D Cummings 1
Affiliation  

Glycan recognition is typically studied using free glycans, but glycopeptide presentations represent more physiological conditions for glycoproteins. To facilitate studies of glycopeptide recognition, we developed Glyco-SPOT synthesis, which enables the parallel production of diverse glycopeptide libraries at microgram scales. The method uses a closed system for prolonged reactions required for coupling Fmoc-protected glycoamino acids, including O-, N-, and S-linked glycosides, and release conditions to prevent side reactions. To optimize reaction conditions and sample reaction progress, we devised a biopsy testing method. We demonstrate the efficient utilization of such microscale glycopeptide libraries to determine the specificity of glycan-recognizing antibodies (e.g., CTD110.6) using microarrays, enzyme specificity on-array and in-solution (e.g., ST6GalNAc1, GCNT1, and T-synthase), and binding kinetics using fluorescence polarization. We demonstrated that the glycosylation on these peptides can be expanded using glycosyltransferases both in-solution and on-array. This technology will promote the discovery of biological functions of peptide modifications by glycans.



中文翻译:

糖肽文库的并行 Glyco-SPOT 合成。

通常使用游离聚糖研究聚糖识别,但糖肽呈现代表糖蛋白的更多生理条件。为了促进糖肽识别的研究,我们开发了 Glyco-SPOT 合成,它能够以微克级并行生产不同的糖肽库。该方法使用封闭系统进行耦合 Fmoc 保护的糖基氨基酸(包括 O-、N-和 S-连接糖苷)所需的长时间反应,并使用释放条件来防止副反应。为了优化反应条件和样品反应进程,我们设计了活检测试方法。我们证明了使用微阵列、阵列上和溶液中的酶特异性(例如。例如,ST6GalNAc1、GCNT1 和 T 合酶),以及使用荧光偏振的结合动力学。我们证明了这些肽上的糖基化可以使用溶液中和阵列上的糖基转移酶进行扩展。该技术将促进聚糖修饰肽的生物学功能的发现。

更新日期:2020-06-30
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