Work

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

Year: 2019

Type: article

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.

Source: Journal of Proteome Research

DOI: 10.1021/acs.jproteome.9b00378

Authors: Benjamin D. Stein, Sébastien Herzig, Salvador Martínez-Bartolomé, Mathieu Lavallée-Adam, Reuben J. Shaw, John R. Yates

Institutions: Scripps Research Institute, Salk Institute for Biological Studies

Language: English

Topic: Advanced Proteomics Techniques and Applications

Subfield: Spectroscopy

Field: Chemistry

Domain: Physical Sciences

Open Access status: green