A PEROXO-Tag Enables Rapid Isolation of Peroxisomes from Human Cells
G. Jordan Ray,
Elizabeth A. Boydston,
Emily Shortt,
Gregory A. Wyant,
Sebastian Lourido,
Walter W. Chen,
David M. Sabatini
Affiliations
G. Jordan Ray
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Department of Biology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; Broad Institute of Harvard and Massachusetts Institute of Technology, 415 Main Street, Cambridge, MA 02142, USA
Elizabeth A. Boydston
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA
Emily Shortt
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA
Gregory A. Wyant
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Department of Biology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; Broad Institute of Harvard and Massachusetts Institute of Technology, 415 Main Street, Cambridge, MA 02142, USA
Sebastian Lourido
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Walter W. Chen
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Department of Biology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; Broad Institute of Harvard and Massachusetts Institute of Technology, 415 Main Street, Cambridge, MA 02142, USA; Corresponding author
David M. Sabatini
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Department of Biology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA; Broad Institute of Harvard and Massachusetts Institute of Technology, 415 Main Street, Cambridge, MA 02142, USA; Corresponding author
Summary: Peroxisomes are metabolic organelles that perform a diverse array of critical functions in human physiology. Traditional isolation methods for peroxisomes can take more than 1 h to complete and can be laborious to implement. To address this, we have now extended our prior work on rapid organellar isolation to peroxisomes via the development of a peroxisomally localized 3XHA epitope tag (“PEROXO-Tag”) and associated immunoprecipitation (“PEROXO-IP”) workflow. Our PEROXO-IP workflow has excellent reproducibility, is easy to implement, and achieves highly rapid (~10 min post homogenization) and specific isolation of human peroxisomes, which we characterize here via proteomic profiling. By offering speed, specificity, reproducibility, and ease of use, the PEROXO-IP workflow should facilitate studies on the biology of peroxisomes.
