Interaction proteomics: characterization of protein complexes using tandem affinity purification-mass spectrometry
Interaction proteomics: characterization of protein complexes using tandem affinity purification-mass spectrometry
复制标题
DOI：
10.1042/bst0380883
复制
发表时间：
2010-08-01
影响因子：
3.9
通讯作者：
Angrand, Pierre-Olivier
中科院分区：
文献类型：
--
作者：
Voelkel, Pamela;Le Faou, Perrine;Angrand, Pierre-Olivier
大多数细胞过程是由组装成多聚体复合物的多种蛋白质执行的。因此，对控制细胞事件的生物途径的精确理解依赖于参与此类多聚体组装的蛋白质的鉴定和生化表征。 MS 的进步使得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质复合物成为可能，而 TAP（串联亲和纯化）方法可以有效地从细胞中分离天然蛋白质复合物以进行蛋白质组学分析。 TAP 是一种基于顺序利用两个亲和标签来纯化蛋白质组装体的通用方法。在第一个纯化步骤中，TAP 标签的蛋白 A 部分与 IgG 珠结合，并通过 TEV（烟草蚀纹病毒）蛋白酶裂解回收与 TAP 标签蛋白相关的蛋白成分。该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，蛋白质复合物通过 TAP 标签的 CBP（钙调蛋白结合肽）固定到钙调蛋白包被的珠子上。 CBP-钙调蛋白相互作用是钙依赖性的，在第二个洗脱步骤中使用钙螯合剂来释放最终的蛋白质复合物制剂，用于通过 MS 进行蛋白质鉴定。事实证明，TAP-MS 方法可以有效地表征细菌、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）的蛋白质复合物。
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.