Etxebarria Juan, Calvo Javier, Martin-Lomas Manuel, Reichardt Niels-Christian
Biofunctional Nanomaterials Unit, San Sebastian, Spain.
ACS Chem Biol. 2012 Oct 19;7(10):1729-37. doi: 10.1021/cb300262x. Epub 2012 Aug 17.
By combining electrophoretic protein separation with lectin-array-based glycan profiling into a single experiment, we have developed a high-throughput method for the rapid analysis of protein glycosylation in biofluids. Fluorescently tagged proteins are separated by SDS-PAGE and transferred by diffusion to a microscope slide covered with multiple copies of 20 different lectins, where they are trapped by specific carbohydrate protein interactions while retaining their relative locations on the gel. A fluorescence scan of the slide then provides an affinity profile with each of the 20 lectins containing a wealth of structural information regarding the present glycans. The affinity of the employed lectins toward N-glycans was verified on a glycan array of 76 structures. While current lectin-based methods for glycan analysis provide only a picture of the bulk glycosylation in complex protein mixtures or are focused on a few specific known biomarkers, our array-based glycoproteomics method can be used as a biomarker discovery tool for the qualitative exploration of protein glycosylation in an unbiased fashion.
通过将蛋白质电泳分离与基于凝集素阵列的聚糖分析整合到一个实验中，我们开发了一种高通量方法，用于快速分析生物流体中的蛋白质糖基化。用荧光标记的蛋白质通过SDS-PAGE进行分离，并通过扩散转移到覆盖有20种不同凝集素多个拷贝的显微镜载玻片上，在那里它们通过特定的碳水化合物-蛋白质相互作用被捕获，同时保留它们在凝胶上的相对位置。然后对载玻片进行荧光扫描，得到与20种凝集素中每一种的亲和力图谱，其中包含有关存在的聚糖的大量结构信息。在所使用的凝集素对N-聚糖的亲和力在一个包含76种结构的聚糖阵列上得到了验证。虽然目前基于凝集素的聚糖分析方法仅提供复杂蛋白质混合物中整体糖基化的情况，或者聚焦于一些特定的已知生物标志物，但我们基于阵列的糖蛋白质组学方法可以用作生物标志物发现工具，以无偏倚的方式定性探索蛋白质糖基化。
