Wellcome Trust Sanger Institute , Wellcome Trust Genome Campus, Hinxton, CB10 1SA Cambridgeshire, United Kingdom.
J Proteome Res. 2012 Mar 2;11(3):1462-74. doi: 10.1021/pr2011632. Epub 2012 Feb 10.
The combination of affinity purification with mass spectrometry analysis has become the method of choice for protein complex characterization. With the improved performance of mass spectrometry technology, the sensitivity of the analyses is increasing, probing deeper into molecular interactions and yielding longer lists of proteins. These identify not only core complex subunits but also the more inaccessible proteins that interact weakly or transiently. Alongside them, contaminant proteins, which are often abundant proteins in the cell, tend to be recovered in affinity experiments because they bind nonspecifically and with low affinity to matrix, tag, and/or antibody. The challenge now lies in discriminating nonspecific binders from true interactors, particularly at the low level and in a larger scale. This review aims to summarize the variety of methods that have been used to distinguish contaminants from specific interactions in the past few years, ranging from manual elimination using heuristic rules to more sophisticated probabilistic scoring approaches. We aim to give awareness on the processing that takes place before an interaction list is reported and on the different types of list curation approaches suited to the different experiments.
亲和纯化与质谱分析相结合已成为蛋白质复合物表征的首选方法。随着质谱技术性能的提高，分析的灵敏度不断提高，可以更深入地探测分子相互作用，并生成更长的蛋白质列表。这些不仅可以鉴定核心复合物亚基，还可以鉴定更难接近的、与基质、标签和/或抗体弱相互作用或瞬时相互作用的蛋白质。与它们一起，污染物蛋白质往往是细胞中丰度较高的蛋白质，由于它们与基质、标签和/或抗体非特异性结合且亲和力低，因此容易在亲和实验中被回收。现在的挑战在于区分非特异性结合物和真正的相互作用物，特别是在低水平和更大规模下。本文综述了过去几年中用于区分特异性相互作用物和污染物的各种方法，从使用启发式规则的手动消除到更复杂的概率评分方法。我们旨在让人们了解在报告相互作用列表之前进行的处理过程，以及适合不同实验的不同类型的列表管理方法。
