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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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。 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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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. 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.