title: Intelligent Mixing of Proteomes for Elimination of False Positives in Affinity Purification-Mass Spectrometry.
abstract: 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.
authors.affiliation: VIB Medical Biotechnology Center , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
authors.forename: Sven
authors.initials: S
authors.lastname: Eyckerman
authors.affiliation: VIB Medical Biotechnology Center , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.|VIB Proteomics Expertise Center , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
authors.forename: Francis
authors.initials: F
authors.lastname: Impens
authors.affiliation: VIB Medical Biotechnology Center , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
authors.forename: Emmy
authors.initials: E
authors.lastname: Van Quickelberghe
authors.affiliation: VIB Medical Biotechnology Center , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
authors.forename: Noortje
authors.initials: N
authors.lastname: Samyn
authors.affiliation: VIB Medical Biotechnology Center , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
authors.forename: Giel
authors.initials: G
authors.lastname: Vandemoortele
authors.affiliation: VIB Medical Biotechnology Center , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
authors.forename: Delphine
authors.initials: D
authors.lastname: De Sutter
authors.affiliation: VIB Medical Biotechnology Center , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Tavernier
authors.affiliation: VIB Medical Biotechnology Center , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.|Department of Biochemistry, Ghent University , Albert Baertsoenkaai 3, B-9000 Ghent, Belgium.
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D002846:Chromatography, Affinity; D007553:Isotope Labeling; D013058:Mass Spectrometry; D025941:Protein Interaction Mapping / Q000379:methods; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D020543:Proteome; C484406:TANK protein, human; C452580:RNF41 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: SILAC; affinity purification-mass spectrometry; protein complex