ISOTOPIC DIFFERENTIATION OF INTERACTIONS AS RANDOM OR TARGETED (I-DIRT)

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 通过亲和标记的蛋白质分离蛋白质复合体为研究生物系统提供了一个强有力的工具，但这项技术经常受到非特异性相互作用蛋白质的共浓缩的影响。我们开发了一种新技术(I-污垢)，在免疫纯化中区分污染物和真正的相互作用，克服了在定义蛋白质复合体方面的这一最具挑战性的问题。这项技术的基本原理是将含有亲和力标记蛋白的同位素轻细胞与不含标记蛋白质的同位素重细胞混合。在分离亲和标记的蛋白质复合体和胰酶消化共富集蛋白质后，通过质谱学鉴定特定相互作用的蛋白质是同位素轻的，而非特异性相互作用的蛋白质看起来是同位素轻和重的混合物。我们已经在J.Proteome Res.(2005)4,1752-6上发表了这项工作，并发表了这项工作的应用(例如，D.J.Lee，S.J.W.Busby，L.F.WestBlade，B.T.Chait免疫分离和I-污垢质谱仪分析大肠杆菌O157：H7 Sakai RNA聚合酶复合体)。J.细菌学190(2008)1284-1289)。 我们继续研究这项技术的更详细的细节，重点是评估具有相对较高离散率的特定交互作用的效果。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Isolation of protein complexes via affinity-tagged proteins provides a powerful tool for studying biological systems, but the technique is often compromised by co-enrichment of non-specifically interacting proteins. We developed a new technique (I-DIRT) that distinguishes contaminants from bona fide interactors in immunopurifications, overcoming this most challenging problem in defining protein complexes. The basic principle of this technique is mixing isotopically light cells that contain an affinity-tagged protein with isotopically heavy cells that do not contain a tagged protein. After isolation of the affinity-tagged protein complex and trypsin digestion of the co-enriching proteins, specifically interacting proteins are identified by mass spectrometry as isotopically light, whilst non-specifically interacting proteins appear as a mixture of isotopically light and heavy. We have published this work in J. Proteome Res. (2005) 4, 1752-6 and have also published applications of this work (e.g., D.J. Lee, S.J.W. Busby, L.F. Westblade, B.T. Chait Immunoisolation and I-DIRT mass spectrometric analysis of the Escherichia coli O157:H7 Sakai RNA polymerase complex. J. Bacteriology 190 (2008) 1284-1289). We continue to investigate the finer details of this technique, with an emphasis on evaluating the effect of specific interactors that have relatively high offrates.