Chromatinomics, Interdisciplinary Research Institute, Université de Lille 1 - Sciences et Technologies/CNRS USR 3078, Parc Scientifique de la Haute Borne, 50 Avenue Halley, F-59658 Villeneuve d'Ascq, France.
Biochem Soc Trans. 2010 Aug;38(4):883-7. doi: 10.1042/BST0380883.
Most cellular processes are carried out by a multitude of proteins that assemble into multimeric complexes. Thus a precise understanding of the biological pathways that control cellular events relies on the identification and on the biochemical characterization of the proteins involved in such multimeric assemblies. Advances in MS have made possible the identification of multisubunit protein complexes isolated from cell lysates with high sensitivity and accuracy, whereas the TAP (tandem affinity purification) methodology efficiently isolates native protein complexes from cells for proteomics analysis. TAP is a generic method based on the sequential utilization of two affinity tags to purify protein assemblies. During the first purification step, the Protein A moiety of the TAP tag is bound to IgG beads, and protein components associated with the TAP-tagged protein are retrieved by TEV (tobacco etch virus) protease cleavage. This enzyme is a sequence-specific protease cleaving a seven-amino-acid recognition site located between the first and second tags. In the second affinity step, the protein complex is immobilized to calmodulin-coated beads via the CBP (calmodulin-binding peptide) of the TAP tag. The CBP-calmodulin interaction is calcium-dependent and calcium-chelating agents are used in the second elution step to release the final protein complex preparation used for protein identification by MS. The TAP-MS approach has proven to efficiently permit the characterization of protein complexes from bacteria, yeast and mammalian cells, as well as from multicellular organisms such as Caenorhabditis elegans, Drosophila and mice.
大多数细胞过程都是由组装成多聚体复合物的多种蛋白质执行的。因此，要精确理解控制细胞事件的生物途径，就需要识别和对参与这种多聚体组装的蛋白质进行生化特性分析。MS 的进步使得能够以高灵敏度和准确性鉴定从细胞裂解物中分离的多亚基蛋白复合物，而 TAP（串联亲和纯化）方法则可有效地从细胞中分离天然蛋白复合物进行蛋白质组学分析。TAP 是一种基于两种亲和标签顺序利用的通用方法，用于纯化蛋白复合物。在第一个纯化步骤中，TAP 标签的 Protein A 部分与 IgG 珠结合，通过 TEV（烟草蚀纹病毒）蛋白酶切割回收与 TAP 标记蛋白相关的蛋白成分。这种酶是一种序列特异性蛋白酶，可切割位于第一和第二标签之间的七个氨基酸识别位点。在第二个亲和步骤中，通过 TAP 标签的 CBP（钙调蛋白结合肽）将蛋白复合物固定在钙调蛋白包被的珠上。CBP-钙调蛋白相互作用是依赖钙的，在第二个洗脱步骤中使用钙螯合剂释放最终的蛋白复合物，用于 MS 进行蛋白鉴定。TAP-MS 方法已被证明可有效地从细菌、酵母和哺乳动物细胞以及多细胞生物（如秀丽隐杆线虫、果蝇和小鼠）中鉴定和表征蛋白复合物。
