Direct and efficient monitoring of glycosyltransferase reactions on gold colloidal nanoparticles by using mass spectrometry
Direct and efficient monitoring of glycosyltransferase reactions on gold colloidal nanoparticles by using mass spectrometry
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DOI：
10.1002/chem.200501267
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发表时间：
2006-08-25
影响因子：
4.3
通讯作者：
Nishimura, Shin-Ichiro
中科院分区：
文献类型：
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作者：
Nagahori, Noriko;Nishimura, Shin-Ichiro
用激光解吸/电离飞行时间质谱仪(LDI-TOF MS)对含有GlcNAc残基的胶体纳米颗粒(GCNPs)上Lewis X三糖的合成进行了研究，建立了一种简单有效的检测GCNPs表面糖基转移酶活性的方法。含有多价糖的GCNPs在水溶液中分散良好，是糖基转移酶反应的良好受体底物。对这些GCNP的直接LDI-TOF MS分析提供了糖衍生物的离子峰，通过S-Au键化学吸附在GCNP上，即使在蛋白质和盐等污染物存在的情况下也是如此。因此，无需纯化即可对少量(0.15亩L)的反应混合物进行MS分析，从而实现了对GCNP上的酶反应的快速直接检测。随后对产物进行了MS/MS分析(LDI-Lift-TOF/TOF方法)，从而确定了通过酶促糖基化作用构建在GCNPs上的糖衍生物的结构。以5‘-二磷酸尿苷(UDP)为抑制剂，建立了糖基转移酶的LDI-TOF-MS定量分析方法。然后将这一简单的方法用于检测产生脑膜炎奈瑟菌β-1，4-半乳糖基转移酶(β-1，4-GalT)的大肠杆菌粗细胞提取物的酶活性。在这种情况下，在MS分析之前，用超滤的方法粗略地纯化GCNP，去除缓冲液和去污剂。GCNP在反应介质中溶解在溶液中，但在纯化过程中是固体的，这对于简单有效地检测粗制生物样品中的酶活性是非常有利的。因此，当结合LDI-TOF MS分析时，含有多种生物分子的GCNPs可能成为快速和直接监测活细胞代谢(代谢组学)的一种通用和有效的工具。
A simple and efficient assay for glycosyltransferase activity on gold colloidal nanoparticles (GCNPs) by using laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) is demonstrated by the enzymatic synthesis of the Lewis X trisaccharide on GCNPs containing GlcNAc residues. GCNPs containing multivalent sugars were well dispersed in aqueous solution and proved to be excellent acceptor substrates for the glycosyltransferase reaction. Direct LDI-TOF MS analysis of these GCNPs provided the ion peaks of the sugar derivatives, chemisorbed through S-Au linkages onto the GCNPs, even in the presence of contaminants such as proteins and salts. Thus, it enabled the rapid and direct detection of the enzymatic reaction on the GCNPs by subjecting a small amount (0.15 mu L) of the reaction mixture to MS analysis without purification. Subsequent MS/MS analyses (LDI-LIFT-TOF/TOF method) of the product-carrying GCNPs enabled the structures of the sugar derivatives that had been constructed on the GCNPs by enzymatic glycosylation to be determined. A quantitative inhibition assay for glycosyltransferase by using LDI-TOF MS analysis on the GCNPs was demonstrated by using uridine 5'-diphosphate (UDP) as the inhibitor. This simple assay was then applied to the detection of the enzymatic activity of a crude cell extract of Escherichia coli, which produces Neisseria meningitidis beta-1,4-galactosyltransferase (beta-1,4-GalT). In this case, the GCNPs were roughly purified by means of ultrafiltration to remove the buffer and detergents before MS analysis. That the GCNPs are dissolved in solution in the reaction medium but are solid in the purification process is greatly advantageous for the simple and efficient detection of enzymatic activity in crude biological samples. Thus, GCNPs containing a variety of biomolecules may become a versatile and efficient tool for the rapid and direct monitoring of metabolism (metabolomics) in living cells when combined with LDI-TOF MS analysis.