High-sensitivity analysis of naturally occurring sugar chains, using a novel fluorescent linker molecule.
High-sensitivity analysis of naturally occurring sugar chains, using a novel fluorescent linker molecule.
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使用新型荧光连接分子对天然存在的糖链进行高灵敏度分析。
DOI：
10.1093/jb/mvp041
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发表时间：
2009
影响因子：
2.7
通讯作者：
Suda,Yasuo
中科院分区：
文献类型：
--
作者：
Sato,Masaki;Ito,Yuji;Arima,Naomichi;Baba,Masanori;Sobel,Michael;Wakao,Masahiro;Suda,Yasuo
表面等离子体共振（SPR）技术是一种实时、非破坏性的检测系统，在分析糖链与蛋白质、病毒和细胞的结合过程中非常方便和有效。该方法的关键是用于将糖链固定到用于SPR的镀金芯片的接头化合物。此外，在分析来自天然来源的微量糖链结构时，精心设计的荧光标记试剂是必不可少的，例如特定细胞表面的糖蛋白。在这份报告中，我们开发了一种新的连接分子，命名为“f-单”，它具有这两个属性：简单的固定化化学和荧光标记。由于该分子含有2，5-二氨基吡啶基和硫辛酸基团，因此可以使用公认的还原胺化反应实现与糖链的缀合。这种糖链和荧光接头的缀合物（荧光配体-缀合物，FLC）具有荧光性质（例如，335 nm，em. 380 nm），并且可以使用具有荧光检测器的HPLC容易地纯化少至1 μg的FLC。FLC的MS和MS/MS分析也是可能的。由于+2 Da较大的MS峰（[M + H + 2]+离子）始终与理论MS峰（[M + H]+）相关（由于硫辛酸部分的还原），因此即使使用未分馏的粗品样品，也很容易发现FLC的MS峰。将FLC固定到金包被的芯片上，并相继完成其随后的SPR分析，如先前使用非荧光配体缀合物进行的那样。
To analyse the binding of sugar chains to proteins, viruses and cells, the surface plasmon resonance (SPR) technique is very convenient and effective because it is a real-time, non-destructive detection system. Key to this method is linker compounds for immobilization of the sugar chains to the gold-coated chip for SPR. Also, well-designed fluorescent labelling reagents are essential when analysing the structure of trace amounts of sugar chains derived from natural sources, such as glycoproteins on the surface of specific cells. In this report, we developed a novel linker molecule, named ‘f-mono’, which has both of these properties: simple immobilization chemistry and a fluorescent label. Since the molecule contains a 2,5-diaminopyridyl group and a thioctic acid group, conjugation with sugar chains can be achieved using the well-established reductive amination reaction. This conjugate of sugar chain and fluorescent linker (fluorescent ligand-conjugate, FLC) has fluorescent properties (ex. 335 nm, em. 380 nm), and as little as 1 μg of FLC can be easily purified using HPLC with a fluorescent detector. MS and MS/MS analysis of the FLC is also possible. As a +2 Da larger MS peak ([M + H + 2]+ion) was always associated with the theoretical MS peak ([M + H]+) (due to the reduction of the thioctic acid moiety), the MS peaks of the FLC were easily found, even using unfractionated crude samples. Immobilization of the FLC onto gold-coated chips, and their subsequent SPR analyses were successively accomplished, as had been performed previously using non-fluorescent ligand conjugates.