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J Proteome Res. 2019 Oct 4;18(10):3703-3714. doi: 10.1021/acs.jproteome.9b00378. Epub 2019 Sep 6.

Comparison of CRISPR Genomic Tagging for Affinity Purification and Endogenous Immunoprecipitation Coupled with Quantitative Mass Spectrometry To Identify the Dynamic AMPKα2 Interactome.

Author information

1
Departments of Molecular Medicine and Neurobiology , The Scripps Research Institute , La Jolla , California , United States.
2
Molecular and Cell Biology Laboratory , The Salk Institute for Biological Studies , La Jolla , California , United States.

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-activated kinase complex AMPK using a catalytic subunit AMPKα2 (PRKAA2 gene) as the bait, based on CRISPR/Cas9-mediated genome editing coupled to stable isotope labeling in cell culture, multidimensional protein identification 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 differences in observed interactors. Additionally, we directly compared each enrichment strategy in the genetically modified cell-line with two separate endogenous antibodies. For each approach, we analyzed the interaction profiles of this protein complex under basal and activated states, and after implementing the same analytical, computational, and statistical analyses, we found that high-confidence protein interactors vary greatly with each method and between commercially available endogenous antibodies.

KEYWORDS:

AMPK; CRISPR tagging; SILAC; affinity purification; dynamic interactions; endogenous immunoprecipitation; interactomics

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