IMixPro: intelligent Mixing of Proteomes for elimination of false positives in affinity purification-mass spectrometry

ABSTRACT: Protein complexes are essential in most organizational and functional aspects of the cell. Different strategies currently exist to analyze 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 out of non-specifically co-purified proteins. Finding the true positive interactors in these lists thus remains highly challenging with proposed solutions on all levels of such experiments. Here, we report an intelligent experimental design based on differential isotopic labeling combined with non-equal 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 where we investigate the interactomes of RAF1, RNF41 and TANK, and also to engineered cell lines expressing epitope-tagged endogenous PTPN14, JIP3 and IQGAP1. For all baits, we observed both known and novel interactions. The results for RNF41 and TANK were compared to a classical affinity purification design which clearly underscores the efficiency and the specificity of the iMixPro approach.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Early Embryonic Cell

DISEASE(S): Disease Free

SUBMITTER: Sven Eyckerman  

LAB HEAD: Kris Gevaert

PROVIDER: PXD004246 | Pride | 2016-09-27

REPOSITORIES: Pride