Intelligent Mixing of Proteomes for Elimination of False Positives in Affinity Purification-Mass Spectrometry

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.