Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia.
J Sep Sci. 2012 Sep;35(18):2445-52. doi: 10.1002/jssc.201200049.
Lectins are capable of recognizing specific glycan structures and serve as invaluable tools for the separation of glycosylated proteins from nonglycosylated proteins in biological samples. We report on the optimization of native multi-lectin affinity chromatography, combining three lectins, namely, concanavalin A, jacalin, and wheat germ agglutinin for fractionation of cellular glycoproteins from MCF-7 breast cancer lysate. We evaluated several conditions for optimum recovery of total proteins and glycoproteins such as low pH and saccharide elution buffers, and the inclusion of detergents in binding and elution buffers. Optimum recovery was observed with overnight incubation of cell lysate with lectins at 4°C, and inclusion of detergent in binding and saccharide elution buffers. Total protein and bound recoveries were 80 and 9%, respectively. Importantly, we found that high saccharide strength elution buffers were not necessary to release bound glycoproteins. This study demonstrates that multi-lectin affinity chromatography can be extended to total cell lysate to investigate the cellular glycoproteome.
凝集素能够识别特定的糖链结构，是分离生物样品中糖基化蛋白和非糖基化蛋白的宝贵工具。我们报告了对天然多凝集素亲和层析的优化，该方法结合了三种凝集素，即伴刀豆球蛋白 A、木菠萝凝集素和麦胚凝集素，用于从 MCF-7 乳腺癌裂解物中分离细胞糖蛋白。我们评估了几种条件以实现总蛋白和糖蛋白的最佳回收，例如低 pH 值和糖洗脱缓冲液，以及在结合和洗脱缓冲液中添加去污剂。在 4°C 下，细胞裂解物与凝集素孵育过夜，并在结合和糖洗脱缓冲液中添加去污剂，可实现最佳回收。总蛋白和结合物的回收率分别为 80%和 9%。重要的是，我们发现释放结合的糖蛋白不需要高糖强度的洗脱缓冲液。这项研究表明，多凝集素亲和层析可以扩展到全细胞裂解物，以研究细胞糖组。
