Carneiro Daniel Gonçalves, Clarke Thomas, Davies Clare C, Bailey Dalan
College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Methods. 2016 Feb 15;95:46-54. doi: 10.1016/j.ymeth.2015.08.022. Epub 2015 Aug 29.
The technological revolution in high-throughput nucleic acid and protein analysis in the last 15 years has launched the field of 'omics' and led to great advances in our understanding of cell biology. Consequently the study of the cellular proteome and protein dynamics, in particular interactomics, has been a matter of intense investigation, specifically the determination and description of complex protein interaction networks in the cell, not only with other proteins but also with RNA and DNA. The analysis of these interactions, beginning with their identification and ultimately resulting in structural level examination, is one of the cornerstones of modern biological science underpinning basic research and impacting on applied biology, biomedicine and drug discovery. In this review we summarise a selection of emerging and established techniques currently being applied in this field with a particular focus on affinity-based purification systems and their optimisation, including tandem affinity purification (TAP) tagging, isolation of proteins on nascent DNA (IPOND) and RNA-protein immunoprecipitation in tandem (RIPiT). The recent application of quantitative proteomics to improve stringency and specificity is also discussed, including the use of metabolic labelling by stable isotope labelling by amino acids in cell culture (SILAC), localization of organelle proteins by isotope tagging (LOPIT) and proximity-dependent biotin identification (BioID). Finally, we describe a range of software resources that can be applied to interactomics, both to handle raw data and also to scrutinise its broader biological context. In this section we focus especially on open-access online interactomic databases such as Reactome and IntAct.
过去15年里高通量核酸和蛋白质分析领域的技术革命开启了“组学”领域,并使我们对细胞生物学的理解取得了巨大进展。因此,细胞蛋白质组和蛋白质动力学的研究,尤其是相互作用组学,一直是深入研究的课题,特别是确定和描述细胞中复杂的蛋白质相互作用网络,不仅包括与其他蛋白质的相互作用,还包括与RNA和DNA的相互作用。对这些相互作用的分析,从识别开始,最终进行结构层面的研究,是现代生物科学的基石之一,支撑着基础研究,并对应用生物学、生物医学和药物发现产生影响。在这篇综述中,我们总结了目前在该领域应用的一系列新兴和成熟技术,特别关注基于亲和的纯化系统及其优化,包括串联亲和纯化(TAP)标签、新生DNA上蛋白质的分离(IPOND)和串联RNA-蛋白质免疫沉淀(RIPiT)。还讨论了定量蛋白质组学在提高严谨性和特异性方面的最新应用,包括细胞培养中氨基酸稳定同位素标记(SILAC)的代谢标记、同位素标记细胞器蛋白质定位(LOPIT)和邻近依赖性生物素识别(BioID)。最后,我们描述了一系列可应用于相互作用组学的软件资源,既用于处理原始数据,也用于审视其更广泛的生物学背景。在本节中,我们特别关注开放获取的在线相互作用组学数据库,如Reactome和IntAct。