Immunoaffinity Purification of Plasma Membrane with Secondary Antibody Superparamagnetic Beads for Proteomic Analysis

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• 作者：Lijun Zhang ; Xi'e Wang ; Xia Peng ; Yingjuan Wei ; Rui Cao ; Zhen Liu ; Jixian Xiong ; Xingfeng Ying ; Ping Chen ; Songping Liang
• 刊名：Journal of Proteome Research
• 出版年：2007
• 出版时间：January 2007
• 年：2007
• 卷：6
• 期：1
• 页码：34 - 43
• 全文大小：480K
• 年卷期：v.6,no.1(January 2007)
• ISSN：1535-3907

文摘

Plasma membrane (PM) has very important roles in cell-cell interaction and signal transduction, andit has been extensively targeted for drug design. A major prerequisite for the analysis of PM proteomeis the preparation of PM with high purity. Density gradient centrifugation has been commonly employedto isolate PM, but it often occurred with contamination of internal membrane. Here we describe a methodfor plasma membrane purification using second antibody superparamagnetic beads that combinessubcellular fractionation and immunoisolation strategies. Four methods of immunoaffinity werecompared, and the variation of crude plasma membrane (CPM), superparamagnetic beads, andantibodies was studied. The optimized method and the number of CPM, beads, and antibodies suitablefor proteome analysis were obtained. The PM of mouse liver was enriched 3-fold in comparison withthe density gradient centrifugation method, and contamination from mitochondria was reduced 2-fold.The PM protein bands were extracted and trypsin-digested, and the resulting peptides were resolvedand characterized by MALDI-TOF-TOF and ESI-Q-TOF, respectively. Mascot software was used to analyzethe data against IPI-mouse protein database. Nonredundant proteins (248) were identified, of which67% are PM or PM-related proteins. No endoplasmic reticulum (ER) or nuclear proteins were identifiedaccording to the GO annotation in the optimized method. Our protocol represents a simple, economic,and reproducible tool for the proteomic characterization of liver plasma membrane.