Van Andel Research Institute, Grand Rapids, Michigan 49503, United States.
Anal Chem. 2013 Feb 5;85(3):1689-98. doi: 10.1021/ac302826a. Epub 2013 Jan 16.
Improved methods for studying glycans could spur significant advances in the understanding and application of glycobiology. The use of affinity reagents such as lectins and glycan-binding antibodies is a valuable complement to methods involving mass spectrometry and chromatography. Many lectins, however, are not useful as analytic tools due to low affinity in vitro. As an approach to increasing lectin avidity to targeted glycans, we tested the use of lectin multimerization. Several biotinylated lectins were linked together through streptavidin interactions. The binding of certain lectins for purified glycoproteins and glycoproteins captured directly out of biological solutions was increased using multimerization, resulting in the detection of lower concentrations of glycoprotein than possible using monomeric detection. The analysis of glycoproteins in plasma samples showed that the level of binding enhancement through multimerization was not equivalent across patient samples. Wheat germ agglutinin (WGA) reactive glycans on fibronectin and thrombospondin-5 were preferentially bound by multimers in pancreatic cancer patient samples relative to control samples, suggesting a cancer-associated change in glycan density that could be detected only through lectin multimerization. This strategy could lead to the more sensitive and informative detection of glycans in biological samples and a broader spectrum of lectins that are useful as analytical reagents.
改进的聚糖研究方法可以促进糖生物学的理解和应用取得重大进展。使用亲和试剂(如凝集素和糖结合抗体)是对涉及质谱和色谱的方法的有益补充。然而,由于体外亲和力低,许多凝集素不能用作分析工具。作为提高针对靶聚糖的凝集素亲合力的一种方法,我们测试了凝集素多聚化的用途。通过链霉亲和素相互作用将几种生物素化的凝集素连接在一起。通过多聚化,某些凝集素与纯化的糖蛋白和直接从生物溶液中捕获的糖蛋白的结合增加,从而可以检测到比使用单体检测更低浓度的糖蛋白。对血浆样本中的糖蛋白的分析表明,通过多聚化增强结合的程度在患者样本之间并不相等。与对照样本相比,多聚体优先结合胰腺癌患者样本中的纤维连接蛋白和血栓反应蛋白-5 上的麦胚凝集素(WGA)反应性聚糖,表明糖密度发生了与癌症相关的变化,只有通过凝集素多聚化才能检测到。该策略可以更灵敏、更全面地检测生物样本中的聚糖,并扩大作为分析试剂有用的凝集素的范围。