In 2012, BioID emerged as a complementary and alternative approach to Y2H and AP–MS 6. Moreover, the protein complexes might be insoluble in standard lysis buffer used in AP–MS. However, weak and transient interactions (such as enzyme–substrate interactions) that do not form stable protein complexes are often difficult to capture using AP–MS owing to their highly dynamic nature. The key advantages of this approach are that it can be applied to large-scale studies and that it shows high intra- and inter-laboratory reproducibility 5.
AP–MS allows the identification of protein interactions under the near physiological conditions within a cell. Once the bait protein interacts with its binding partners, the protein complex forms and can be purified from the cell lysate using a matrix that specifically recognizes the affinity tag. In contrast, AP depends on use of antibodies or, more commonly, on the expression of a bait protein with an affinity tag in cells. The main drawbacks of the Y2H are the possibility of a high number of false-positive and false-negative interactions. It is an affordable method that requires minimal equipment and can be carried out in most laboratories 2, 3, 4. The Y2H assay is an in vivo genetic method to screen for binary PPIs 1. Three methods commonly used for the systematic identification of PPIs are yeast two-hybrid (Y2H) assays, AP–MS and proximity-dependent BioID. The entire procedure can be completed within 25 d.Īlmost all biological processes are mediated through protein–protein interactions (PPIs) hence, the accurate identification and annotation of PPIs is of great importance in systems biology. In this protocol, we present the detailed three-stage procedure for the MAC-tag workflow: (1) cell line generation for the MAC-tagged POI (2) parallel AP–MS and BioID protein purification followed by MS analysis and (3) protein interaction data analysis, data filtration and visualization with our localization visualization platform.
This localization database is accessible via our online platform ( ) to predict the cellular localization of a protein of interest (POI) depending on its identified interactors. We have applied the MAC-tag workflow to a selection of subcellular markers to provide a global view of the cellular protein interactome landscape. Therefore, we developed the Multiple Approaches Combined (MAC)-tag workflow, which allows for both AP–MS and BioID analysis with a single construct and with almost identical protein purification and mass spectrometry (MS) identification procedures.
Integration of AP–MS and BioID data has been shown to comprehensively characterize a protein’s molecular context, but interactome analysis using both methods in parallel is still labor and resource intense with respect to cell line generation and protein purification. Whereas AP−MS results in the identification of proteins that are in a stable complex, BioID labels and identifies proteins that are in close proximity to the bait, resulting in overlapping yet distinct protein identifications. Affinity purification coupled with mass spectrometry (AP–MS) and proximity-dependent biotinylation identification (BioID) methods have made substantial contributions to interaction proteomics studies.
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