Abstract
Protein-protein interactions govern almost all cellular functions. These complex networks of stable and transient associations can be mapped by affinity purification mass spectrometry (AP-MS) and complementary proximity-based labeling methods such as BioID. To exploit the advantages of both strategies, we here design and optimize an integrated approach combining AP-MS and BioID in a single construct, which we term MAC-tag. We systematically apply the MAC-tag approach to 18 subcellular and 3 sub-organelle localization markers, generating a molecular context database, which can be used to define a protein’s molecular location. In addition, we show that combining the AP-MS and BioID results makes it possible to obtain interaction distances within a protein complex. Taken together, our integrated strategy enables the comprehensive mapping of the physical and functional interactions of proteins, defining their molecular context and improving our understanding of the cellular interactome.
Highlights
Protein-protein interactions govern almost all cellular functions
In addition to optimizing the experimental steps, we focused on the compatibility of the two methods and to the simplicity of the analysis pipelines to generate a process with improved performance and reproducibility on detecting protein-protein interactions
This workflow features state-of-the-art affinity purification using Strep-tag to identify and quantify protein-protein interaction and protein complex stoichiometry; identification of transient or close-proximity interactions with BioID; visualization of the bait protein and the proximal interactors with immunofluorescence microscopy; and defining the molecular context with mass spectrometry (MS) microscopy utilizing the reference dataset obtained by identifying proximal interactors for bona fide subcellular localization markers
Summary
Protein-protein interactions govern almost all cellular functions These complex networks of stable and transient associations can be mapped by affinity purification mass spectrometry (AP-MS) and complementary proximity-based labeling methods such as BioID. The AP-MS approach relies on expression of a bait protein coupled with an epitope tag or antibodies targeting the endogenous bait protein, allowing purification of the bait protein together with the associating proteins (preys) This approach has been proven well suited for even large-scale high-throughput studies, and to yield highly reproducible data in both intra- and inter-laboratory usage[2]. The HA epitope facilitates additional followup approaches such as ChIP-Seq[17] and purification of the crosslinked proteins for cross-linking coupled with mass spectrometry (XL-MS)[18], making the MAC-tag almost as versatile as the Swiss Army knife
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