Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast
Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast
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DOI：
10.1074/mcp.m300099-mcp200
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发表时间：
2004-03-01
影响因子：
7
通讯作者：
Deshaies, RJ
中科院分区：
文献类型：
--
作者：
Graumann, J;Dunipace, LA;Deshaies, RJ
一种被称为“MudPIT”的多维色谱-质谱学相结合的方法能够快速识别与标记诱饵相互作用的蛋白质，同时绕过了与分析从十二烷基硫酸钠-聚丙烯酰胺凝胶中提取的多肽相关的一些问题。然而，MudPIT的重复性、成功率和对数十种蛋白质的快速鉴定的适用性尚未见报道。我们在这里展示了MudPIT重复性地为发芽酵母Gcn5p确定了真正的合作伙伴。此外，我们成功地应用MudPIT通过一组标记多肽进行快速筛选，以确定新的蛋白质相互作用。用一种新的串联亲和纯化(TAP)标签修饰了25种通过有丝分裂参与转录和进程的蛋白质。TAP-MudPIT分析了22个表达这些标记蛋白的酵母菌株，发现了21个诱饵的已知或可能的相互作用伙伴，这个数字与传统方法相比是有利的。这里确定的蛋白质包括102个已知的和279个潜在的物理相互作用。即使对于被深入研究的Swi2p/Snf2p，即Swi/SNF染色质重塑复合体的催化亚单位，我们的分析也发现了一种新的相互作用蛋白，Rtt102p。对Rtt102p的相互标记和TAP-MudPIT分析揭示了Swi/Snf和RSC复合体的亚基，确定Rtt102p是这些染色质重塑机器的共同相互作用者，也可能是染色质重塑机器的组成部分。我们的经验表明，研究人员在传统的分子/细胞生物学实验室中使用单离子陷阱仪器，通过系统地应用TAP-MudPIT来对途径、细胞器或过程进行蛋白质组学表征(即“途径蛋白质组学”)是可行的。
A combined multidimensional chromatography- mass spectrometry approach known as " MudPIT" enables rapid identification of proteins that interact with a tagged bait while bypassing some of the problems associated with analysis of polypeptides excised from SDS- polyacrylamide gels. However, the reproducibility, success rate, and applicability of MudPIT to the rapid characterization of dozens of proteins have not been reported. We show here that MudPIT reproducibly identified bona fide partners for budding yeast Gcn5p. Additionally, we successfully applied MudPIT to rapidly screen through a collection of tagged polypeptides to identify new protein interactions. Twenty- five proteins involved in transcription and progression through mitosis were modified with a new tandem affinity purification ( TAP) tag. TAP- MudPIT analysis of 22 yeast strains that expressed these tagged proteins uncovered known or likely interacting partners for 21 of the baits, a figure that compares favorably with traditional approaches. The proteins identified here comprised 102 previously known and 279 potential physical interactions. Even for the intensively studied Swi2p/ Snf2p, the catalytic subunit of the Swi/ Snf chromatin remodeling complex, our analysis uncovered a new interacting protein, Rtt102p. Reciprocal tagging and TAP- MudPIT analysis of Rtt102p revealed subunits of both the Swi/ Snf and RSC complexes, identifying Rtt102p as a common interactor with, and possible integral component of, these chromatin remodeling machines. Our experience indicates it is feasible for an investigator working with a single ion trap instrument in a conventional molecular/ cellular biology laboratory to carry out proteomic characterization of a pathway, organelle, or process ( i. e. " pathway proteomics") by systematic application of TAP- MudPIT.