Nat Cell Biol 13, 1016C1023 (2011). 2. NIHMS1607578-supplement-Supplemental_Number_2.pdf (1.0M) GUID:?164ABC95-11FF-4A9D-A3BA-266303C448BD Abstract Recent advances in genome editing technologies have enabled the insertion of epitope tags at endogenous loci with relative efficiency. We describe an approach for investigation of protein interaction dynamics of the AMP triggered kinase complex HA15 AMPK using a catalytic subunit AMPK2 (gene) as the bait, based on CRISPR/Cas9-mediated genome editing coupled to Stable Isotope Labeling in Cell tradition, Multidimensional Protein Recognition Technology and computational and statistical analyses. Furthermore, we directly compare this genetic epitope-tagging approach to endogenous immunoprecipitations of the same gene under homologous conditions to assess variations in observed interactors. Additionally we directly compared each enrichment strategy in the genetically revised cell-line with 2 independent endogenous antibodies. For each approach, we analyzed the interaction profiles of this protein complex under basal and triggered claims, and after implementing the same analytical, computational and statistical analyses, we found that high-confidence protein interactors vary greatly with each method and HA15 between commercially available endogenous antibodies. Keywords: CRISPR-tagging, Interactomics, Dynamic relationships, SILAC, AMPK, Affintiy-Purification, Endogenous Immunoprecipitation Intro. Proteins often come together in cells to form complexes that can transmit signals, perform functions or to create higher order structures. As a means to identify the components of these complexes, a number of strategies have been used to enrich proteins in complexes including co-elution, co-sedimentation, affinity connection and immunoprecipitation (IP) 1. The process to identify proteins in complexes often begins with enrichment of proteins by IP, followed by western blotting to identify if a candidate protein is present 1. The introduction of mass spectrometry centered methods to determine all proteins enriched through an IP or additional methods produced an unbiased and more comprehensive approach to the analysis of protein complexes 2-4. However, while all proteins present in an IP can be recognized using mass spectrometry, the careful use of settings, replicates and statistics to separate transmission from noise in the data is definitely necessary. Immuno- or affinity precipitation methods have proven to be the highest resolving methods for enrichment of protein complexes, therefore providing a means to understand protein networks traveling physiology. However, immunoprecipitation methods require a high-quality antibody specific to a bait protein, so large-scale analysis of protein complexes would require antibodies for many different bait proteins. While you will find antibodies for many proteins, the collection is definitely sporadic and far from complete 5. More importantly, the quality of antibodies can vary greatly. In 1988 epitope tags were first launched into proteins as a means to create a common handle for protein purification Rabbit Polyclonal to ADA2L and capture of interacting proteins 6-8. Epitope tagging strategies have provided a means to perform large-scale protein interaction studies. The Tandem Affinity Purification (Faucet) tag is an example of a high affinity tag that can be used to enrich for the core components of a protein complex 9, but the two-step Faucet purification process can result in the loss of low affinity interactors during the extra enrichment step. A powerful feature of the Faucet system in candida is the ability to use homologous recombination as a means to directly place the tag into a gene, which would then become indicated at endogenous levels. Gavin et al used the TAP system inside a large-scale analysis of the candida interactome 10, but for many biological systems homologous recombination is definitely inefficient and thus not a viable option to introduce epitope HA15 tag sequences into genes. An alternate approach uses transiently overexpressed proteins from plasmids to incorporate an epitope tag 11-15. This approach can be used to express proteins that may not be indicated under laboratory conditions, thus permitting better capture of low affinity interactors because of higher expression levels, but it can also result in a higher level of noise based on mis-localization of the protein or perturbation of the complexs stoichiometry. Additionally, an exogenously HA15 indicated epitope tagged protein competes with the endogenously indicated protein for interactors unless it is removed or not indicated.11 Ho et al used an exogenously overexpressed FLAG tag system to analyze the yeast interactome. A comparison of the two large-scale candida interactome studies (von Mering et al) showed distinct variations in protection and accuracy HA15 between the Gavin et al and Ho et al results, with the Gavin et al study yielding more accurate results (based on current knowledge.