Chang Ing-Feng
Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557, USA.
Proteomics. 2006 Dec;6(23):6158-66. doi: 10.1002/pmic.200600225.
In recent years, MS has been widely used to study protein complex in eukaryotes. The identification of interacting proteins of a particular target protein may help defining protein-protein interaction and proteins of unknown functions. To isolate protein complexes, high-speed ultracentrifugation, sucrose density-gradient centrifugation, and coimmunoprecipitation have been widely used. However, the probability of getting nonspecific binding is comparatively high. Alternatively, by use of one- or two-step (tandem affinity purification) epitope-tag affinity purification, protein complexes can be isolated by affinity or immunoaffinity columns. These epitope-tags include protein A, hexahistidine (His), c-Myc, hemaglutinin (HA), calmodulin-binding protein, FLAG, maltose-binding protein, Strep, etc. The isolated protein complex can then be subjected to protease (i.e., trypsin) digestion followed by an MS analysis for protein identification. An example, the epitope-tag purification of the Arabidopsis cytosolic ribosomes, is addressed in this article to show the success of the application. Several representative protein complexes in eukaryotes been isolated and characterized by use of this approach are listed. In this review, the comparison among different tag systems, validation of interacting relationship, and choices of MS analysis method are addressed. The successful rate, advantages, limitations, and challenges of the epitope-tag purification are also discussed.
近年来，质谱（MS）已被广泛用于研究真核生物中的蛋白质复合物。鉴定特定靶蛋白的相互作用蛋白可能有助于确定蛋白质-蛋白质相互作用以及未知功能的蛋白质。为了分离蛋白质复合物，高速超速离心、蔗糖密度梯度离心和免疫共沉淀已被广泛应用。然而，非特异性结合的概率相对较高。另外，通过使用一步或两步（串联亲和纯化）表位标签亲和纯化，可以通过亲和或免疫亲和柱分离蛋白质复合物。这些表位标签包括蛋白A、六组氨酸（His）、c-Myc、血凝素（HA）、钙调蛋白结合蛋白、FLAG、麦芽糖结合蛋白、链霉亲和素等。然后可以对分离的蛋白质复合物进行蛋白酶（即胰蛋白酶）消化，随后进行质谱分析以鉴定蛋白质。本文以拟南芥胞质核糖体的表位标签纯化为例，展示了该应用的成功。列出了几种通过使用这种方法分离和表征的真核生物中具有代表性的蛋白质复合物。在这篇综述中，讨论了不同标签系统之间的比较、相互作用关系的验证以及质谱分析方法的选择。还讨论了表位标签纯化的成功率、优点、局限性和挑战。
