Lee Yu-Chen, Srajer Gajdosik Martina, Josic Djuro, Lin Sue-Hwa
Department of Molecular Pathology, University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Methods Mol Biol. 2012;909:29-41. doi: 10.1007/978-1-61779-959-4_3.
Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.
分离高度纯化的质膜是构建质膜蛋白质组的关键步骤。传统的质膜分离方法利用了细胞器的密度差异。虽然差速离心法足以富集质膜，但该过程冗长，且膜组分的回收率较低。重要的是，质膜会被其他细胞器严重污染。传统的琼脂糖亲和基质适用于分离蛋白质，但由于琼脂糖的密度，在分离细胞器方面存在局限性。将亲和配体固定在磁珠上可通过磁铁将亲和基质与细胞器分离，可用于开发细胞器分离方法。我们开发了一种使用凝集素伴刀豆球蛋白A（ConA）磁珠分离质膜的简单方法。通过将生物素化的ConA与链霉亲和素磁珠结合，将ConA固定在磁珠上。ConA磁珠用于结合膜中存在的糖基化蛋白质。用含有竞争性糖α-甲基甘露糖苷的去污剂将结合的膜从磁珠上溶解下来。在本研究中，我们描述了按照内维尔所述使用蔗糖密度梯度离心法分离大鼠肝脏质膜的步骤。然后我们使用ConA磁珠进一步纯化膜组分。经过这一纯化步骤后，主要的肝脏质膜蛋白质，尤其是高度糖基化的蛋白质和含有跨膜结构域的蛋白质，可通过液相色谱-电喷雾串联质谱（LC-ESI-MS/MS）进行鉴定。虽然此处未作描述，但磁珠法也可用于从细胞裂解物中分离质膜。这种膜纯化方法应能加快质膜蛋白质组的编目。
