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Free keywords:
NUCLEAR-PORE COMPLEX, SACCHAROMYCES-CEREVISIAE, QUANTITATIVE PROTEOMICS,
MACROMOLECULAR COMPLEXES, BAC TRANSGENEOMICS, YEAST, QUANTIFICATION,
IDENTIFICATION, MIXTURES, SYSTEMChemistry;
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.
