Alberta Glycomics Centre and Department of Chemistry, University of Alberta , Edmonton, Alberta, Canada , T6G 2G2.
Anal Chem. 2016 Aug 16;88(16):8224-31. doi: 10.1021/acs.analchem.6b02044. Epub 2016 Jul 29.
An electrospray ionization mass spectrometry (ESI-MS) assay for screening carbohydrate libraries against lectins is described. The assay is based on the proxy protein ESI-MS method, which combines direct ESI-MS protein-ligand binding measurements and competitive protein binding, to simultaneously detect and quantify protein-carbohydrate interactions. Specific interactions between components of the library and the target protein (PT) are identified from changes in the relative abundances (as measured by ESI-MS) of the carbohydrate complexes of a proxy protein (Pproxy), which binds to all components of the library with known affinity, upon addition of PT to the solution. The magnitude of the change in relative abundance of a given Pproxy-ligand complex provides a quantitative measure of the affinity of the corresponding PT-ligand interaction. A mathematical framework for the implementation of the method in the case of monovalent (single binding site) Pproxy and monovalent and multivalent (multiple equivalent and independent binding sites) PT is described. The application of the method to screen small libraries of oligosaccharides, on the basis of human histo-blood group antigens and milk oligosaccharides, against an N-terminal fragment of the family 51 carbohydrate-binding module, a fucose-binding lectin from Ralstonia solanacearum, and human norovirus VA387 P particle (24-mer of the protruding domain of the capsid protein), serves to demonstrate the reliability and versatility of the assay.
本文描述了一种用于筛选糖库与凝集素相互作用的电喷雾电离质谱（ESI-MS）分析方法。该方法基于“替代蛋白 ESI-MS”方法，将直接 ESI-MS 蛋白-配体结合测量和竞争性蛋白结合相结合，以同时检测和定量蛋白-碳水化合物相互作用。通过结合已知亲和力的替代蛋白（Pproxy）与库中所有成分的结合，在向溶液中加入目标蛋白（PT）后，通过检测替代蛋白的碳水化合物复合物的相对丰度（通过 ESI-MS 测量）的变化，确定库中各成分与目标蛋白之间的特异性相互作用。特定 Pproxy-配体复合物的相对丰度变化的幅度提供了对应 PT-配体相互作用亲和力的定量度量。本文描述了在单价（单结合位点）替代蛋白 Pproxy 和单价和多价（多个等效和独立结合位点）PT 的情况下，实施该方法的数学框架。该方法已应用于基于人组织血型抗原和乳寡糖的小型寡糖库的筛选，针对家族 51 碳水化合物结合模块的 N 末端片段、来自茄科雷尔氏菌的岩藻糖结合凝集素以及人诺如病毒 VA387 P 颗粒（衣壳蛋白突出结构域的 24 个残基）进行了验证，证明了该方法的可靠性和通用性。
