Department of Chemistry and Biomolecular Sciences, Macquarie University, Australia.
OMICS. 2010 Aug;14(4):487-99. doi: 10.1089/omi.2010.0075.
One common method used for analyzing the glycoproteome is chromatography using multiple lectins that display different affinities toward oligosaccharide structures. Much has been done to determine lectin affinity using standard glycoproteins with known glycosylation; however, a knowledge of the selectivity and specificity of lectins exposed to complex mixtures of proteins is required if they are to be used as a means of studying the glycoproteome. In the present study, three lectins (Concanavalin A, Jacalin, and Wheat Germ Agglutinin) were used to fractionate glycoproteins from two different complex environments: (1) cell membranes and (2) plasma. Reproducible enrichment of glycoproteins from these samples has been shown to result from the combined use of these lectins. However, the global glycan profiles of the released N- and O-linked oligosaccharides from the glycoproteins retained by the lectins, and from those glycoproteins that did not bind, using both these complex samples, were found to be very similar. That is, although the lectins selectively and reproducibly retained some glycoproteins, other proteins with the same attached oligosaccharide structures did not bind. Some small N- and O-glycan differences were observed in the bound fractions but there was little absolute specificity toward individual oligosaccharide structures known to have high affinity to these lectins. These data indicate that lectins are useful for fractionating glycoproteins from complex mixtures, but that the overall glycoproteome is not isolated by this approach.
一种常用于分析糖蛋白组的常用方法是使用多种具有不同寡糖结构亲和力的凝集素来进行色谱分析。已经做了很多工作来使用具有已知糖基化的标准糖蛋白来确定凝集素的亲和力；然而，如果要将凝集素用作研究糖蛋白组的手段，则需要了解暴露于复杂蛋白质混合物中的凝集素的选择性和特异性。在本研究中，使用三种凝集素（刀豆球蛋白 A、木菠萝凝集素和麦胚凝集素）从两种不同的复杂环境中分离糖蛋白：（1）细胞膜和（2）血浆。从这些样品中得到的糖蛋白的可重现性富集表明，这些凝集素的联合使用是有效的。然而，从糖蛋白保留的凝集素释放的 N-和 O-连接的寡糖的全局聚糖图谱，以及从那些不结合的糖蛋白中，使用这两种复杂的样品，发现非常相似。也就是说，尽管凝集素选择性和可重复地保留了一些糖蛋白，但其他具有相同附着的寡糖结构的蛋白质没有结合。在结合的部分中观察到一些小的 N-和 O-糖基化差异，但对于已知与这些凝集素有高亲和力的个别寡糖结构几乎没有绝对特异性。这些数据表明，凝集素可用于从复杂混合物中分离糖蛋白，但通过这种方法并不能完全分离整个糖蛋白组。
