Wei Xin, Dulberger Charles, Li Lingjun
Department of Chemistry and School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705.
Anal Chem. 2010 Aug 1;82(15):6329-6333. doi: 10.1021/ac1004844.
Membrane glycoproteins play vital roles in many fundamental physiological and pathophysiological processes in the central nervous system and represent important targets for pharmaceuticals and biomarker discovery. However, their isolation and characterization have been greatly limited. Lectin affinity chromatography (LAC) has evolved as a powerful method to enrich glycoproteins in biofluid and cell/tissue lysate. However, its use in the hydrophobic fraction of the samples has rarely been explored. In this study, we have conducted a systematic investigation on the lectin binding efficiency in the presence of four commonly used detergents. We have found that, under certain concentrations, detergents can minimize nonspecific binding and facilitate the elution of hydrophobic glycoproteins. With the detergent assisted lectin affinity chromatography (DALAC), a total of 1491 proteins were identified with low numbers of false positives from two lectins. Proteins (699) were identified with at least two unique peptides, of which 219 are membrane glycoproteins. Compared to the traditional methods, the DALAC approach significantly increased the recovery of plasma membrane and glycoproteins. NP-40 is recommended as a well rounded detergent for DALAC, but the conditions for enriching certain target proteins need to be empirically determined. This study represents the first global identification of the murine brain glycoproteome.
膜糖蛋白在中枢神经系统的许多基本生理和病理生理过程中发挥着至关重要的作用，是药物研发和生物标志物发现的重要靶点。然而，它们的分离和表征受到很大限制。凝集素亲和色谱法（LAC）已发展成为一种在生物流体和细胞/组织裂解物中富集糖蛋白的强大方法。然而，其在样品疏水部分的应用很少被探索。在本研究中，我们对四种常用去污剂存在下的凝集素结合效率进行了系统研究。我们发现，在一定浓度下，去污剂可以最小化非特异性结合，并促进疏水糖蛋白的洗脱。通过去污剂辅助凝集素亲和色谱法（DALAC），从两种凝集素中鉴定出总共1491种蛋白质，假阳性数量较少。鉴定出至少有两个独特肽段的蛋白质（699种），其中219种是膜糖蛋白。与传统方法相比，DALAC方法显著提高了质膜和糖蛋白的回收率。推荐NP - 40作为DALAC的通用去污剂，但富集某些目标蛋白的条件需要通过实验确定。本研究首次对小鼠脑糖蛋白组进行了全面鉴定。
