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

Overview

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.

publication date

  • September 6, 2019

Research

keywords

  • AMP-Activated Protein Kinases
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Genomics
  • Protein Interaction Mapping

Identity

PubMed Central ID

  • PMC7512655

Scopus Document Identifier

  • 85072692551

Digital Object Identifier (DOI)

  • 10.1172/Jci13505

PubMed ID

  • 31398040

Additional Document Info

volume

  • 18

issue

  • 10