title: Accurate protein complex retrieval by affinity enrichment mass spectrometry (AE-MS) rather than affinity purification mass spectrometry (AP-MS).
abstract: Protein-protein interactions are fundamental to the understanding of biological processes. Affinity purification coupled to mass spectrometry (AP-MS) is one of the most promising methods for their investigation. Previously, complexes were purified as much as possible, frequently followed by identification of individual gel bands. However, todays mass spectrometers are highly sensitive, and powerful quantitative proteomics strategies are available to distinguish true interactors from background binders. Here we describe a high performance affinity enrichment-mass spectrometry method for investigating protein-protein interactions, in which no attempt at purifying complexes to homogeneity is made. Instead, we developed analysis methods that take advantage of specific enrichment of interactors in the context of a large amount of unspecific background binders. We perform single-step affinity enrichment of endogenously expressed GFP-tagged proteins and their interactors in budding yeast, followed by single-run, intensity-based label-free quantitative LC-MS/MS analysis. Each pull-down contains around 2000 background binders, which are reinterpreted from troubling contaminants to crucial elements in a novel data analysis strategy. First the background serves for accurate normalization. Second, interacting proteins are not identified by comparison to a single untagged control strain, but instead to the other tagged strains. Third, potential interactors are further validated by their intensity profiles across all samples. We demonstrate the power of our AE-MS method using several well-known and challenging yeast complexes of various abundances. AE-MS is not only highly efficient and robust, but also cost effective, broadly applicable, and can be performed in any laboratory with access to high-resolution mass spectrometers.
authors.affiliation: From the ‡Department Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Eva C
authors.initials: EC
authors.lastname: Keilhauer
authors.affiliation: From the ‡Department Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Marco Y
authors.initials: MY
authors.lastname: Hein
authors.affiliation: From the ‡Department Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany mmann@biochem.mpg.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
mesh_terms: D002853:Chromatography, Liquid; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000737:chemistry
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins