Tandem detergent-extraction and immunoprecipitation of proteinopathy: Scalable enrichment of ALS-associated TDP-43 aggregates
Baggio A. Evangelista,
Shannon R. Cahalan,
Joey V. Ragusa,
Angie Mordant,
Julie C. Necarsulmer,
Robert J. Perna,
Tejazaditya Ajit,
Kristen White,
Natalie K. Barker,
Xu Tian,
Sarah Cohen,
Rick Meeker,
Laura E. Herring,
Todd J. Cohen
Affiliations
Baggio A. Evangelista
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Shannon R. Cahalan
Medical Student Training in Aging Research, Center for Aging and Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Joey V. Ragusa
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Angie Mordant
Proteomics Core Facility, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Julie C. Necarsulmer
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Robert J. Perna
Department of Neurology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Tejazaditya Ajit
Department of Neurology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Kristen White
Microscopy Services Laboratory, Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Natalie K. Barker
Proteomics Core Facility, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Xu Tian
Department of Neurology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Sarah Cohen
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Rick Meeker
Department of Neurology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Laura E. Herring
Proteomics Core Facility, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Todd J. Cohen
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Neurology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; UNC Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Corresponding author
Summary: Transactive response DNA-binding protein of 43 kDa (TDP-43) is a highly conserved, ubiquitously expressed nucleic acid-binding protein that regulates DNA/RNA metabolism. Genetics and neuropathology studies have linked TDP-43 to several neuromuscular and neurological disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Under pathological conditions, TDP-43 mislocalizes to the cytoplasm where it forms insoluble, hyper-phosphorylated aggregates during disease progression. Here, we optimized a scalable in vitro immuno-purification strategy referred to as tandem detergent-extraction and immunoprecipitation of proteinopathy (TDiP) to isolate TDP-43 aggregates that recapitulate those identified in postmortem ALS tissue. Moreover, we demonstrate that these purified aggregates can be utilized in biochemical, proteomics, and live-cell assays. This platform offers a rapid, accessible, and streamlined approach to study ALS disease mechanisms, while overcoming many limitations that have hampered TDP-43 disease modeling and therapeutic drug discovery efforts.
