Eyckerman Sven, Impens Francis, Van Quickelberghe Emmy, Samyn Noortje, Vandemoortele Giel, De Sutter Delphine, Tavernier Jan, Gevaert Kris
VIB Medical Biotechnology Center , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
Department of Biochemistry, Ghent University , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
J Proteome Res. 2016 Oct 7;15(10):3929-3937. doi: 10.1021/acs.jproteome.6b00517. Epub 2016 Sep 28.
Protein complexes are essential in all organizational and functional aspects of the cell. Different strategies currently exist for analyzing such protein complexes by mass spectrometry, including affinity purification (AP-MS) and proximal labeling-based strategies. However, the high sensitivity of current mass spectrometers typically results in extensive protein lists mainly consisting of nonspecifically copurified proteins. Finding the true positive interactors in these lists remains highly challenging. Here, we report a powerful design based on differential labeling with stable isotopes combined with nonequal mixing of control and experimental samples to discover bona fide interaction partners in AP-MS experiments. We apply this intelligent mixing of proteomes (iMixPro) concept to overexpression experiments for RAF1, RNF41, and TANK and also to engineered cell lines expressing epitope-tagged endogenous PTPN14, JIP3, and IQGAP1. For all baits, we confirmed known interactions and found a number of novel interactions. The results for RNF41 and TANK were compared to a classical affinity purification experiment, which demonstrated the efficiency and specificity of the iMixPro approach.
蛋白质复合物在细胞的所有组织和功能方面都至关重要。目前存在不同的通过质谱分析此类蛋白质复合物的策略，包括亲和纯化（AP-MS）和基于邻近标记的策略。然而，当前质谱仪的高灵敏度通常会产生大量主要由非特异性共纯化蛋白质组成的蛋白质列表。在这些列表中找到真正的阳性相互作用蛋白仍然极具挑战性。在此，我们报告了一种强大的设计，该设计基于稳定同位素的差异标记，并结合对照和实验样品的不等量混合，以在AP-MS实验中发现真正的相互作用伙伴。我们将这种蛋白质组的智能混合（iMixPro）概念应用于RAF1、RNF41和TANK的过表达实验，以及应用于表达表位标记的内源性PTPN14、JIP3和IQGAP1的工程细胞系。对于所有诱饵，我们证实了已知的相互作用，并发现了许多新的相互作用。将RNF41和TANK的结果与经典亲和纯化实验进行了比较，这证明了iMixPro方法的效率和特异性。
