Quantitative proteomics reveals regulation of dynamic components within TATA-binding protein (TBP) transcription complexes
Quantitative proteomics reveals regulation of dynamic components within TATA-binding protein (TBP) transcription complexes
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DOI：
10.1074/mcp.m700306-mcp200
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发表时间：
2008-05-01
影响因子：
7
通讯作者：
Heck, Albert J. R.
中科院分区：
文献类型：
--
作者：
Mousson, Florence;Kolkman, Annemieke;Heck, Albert J. R.
亲和纯化结合同位素标记的蛋白质已被证明是一个强大的方法来区分特异性和非特异性的相互作用。然而，在标准SILAC（细胞培养物中氨基酸的稳定同位素标记）方法中，动态组分可能很容易被指定为非特异性。我们比较了两种亲和纯化方案，其结合揭示了蛋白质复合物的动力学信息。我们集中在真核转录的核心组成部分，人类TATA结合蛋白，这是参与不同的复合物。所有已知的TATA结合蛋白相关因子（TAFs）被检测为特定的相互作用。有趣的是，其中之一，BTAF1，在亲和纯化过程中，在细胞提取物中显着交换。其他TAF未显示此行为。细胞周期同步化表明BTAF1交换在有丝分裂过程中受到调节。这两种亲和纯化蛋白质的组合允许定量方法来鉴定任何蛋白质复合物中的瞬时组分。
Affinity purification in combination with isotope labeling of proteins has proven to be a powerful method to discriminate specific from nonspecific interactors. However, in the standard SILAC (stable isotope labeling by amino acids in cell culture) approach dynamic components may easily be assigned as nonspecific. We compared two affinity purification protocols, which in combination revealed information on the dynamics of protein complexes. We focused on the central component in eukaryotic transcription, the human TATA-binding protein, which is involved in different complexes. All known TATA-binding protein-associated factors (TAFs) were detected as specific interactors. Interestingly one of them, BTAF1, exchanged significantly in cell extracts during the affinity purification. The other TAFs did not display this behavior. Cell cycle synchronization showed that BTAF1 exchange was regulated during mitosis. The combination of the two affinity purification proto cols allows a quantitative approach to identify transient components in any protein complex.