Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2
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Ting_Spatially resolved.pdf
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Author(s)
Hung, Victoria
Udeshi, Namrata D.
Lam, Stephanie Shih-Min
Loh, Ken H.
Cox, Kurt James
Pedram, Kayvon
Carr, Steven A.
Ting, Alice Y.
Date Issued
February 2016
Journal
Nature Protocols
Publisher
Nature Publishing Group
Citation
Hung, Victoria, Namrata D. Udeshi, Stephanie S. Lam, Ken H. Loh, Kurt J. Cox, Kayvon Pedram, Steven A. Carr, and Alice Y. Ting. "Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2." Nature Protocols 11:3 (February 2016), pp.456-475.
Version
Author's final manuscript
Abstract
This protocol describes a method to obtain spatially resolved proteomic maps of specific compartments within living mammalian cells. An engineered peroxidase, APEX2, is genetically targeted to a cellular region of interest. Upon the addition of hydrogen peroxide for 1 min to cells preloaded with a biotin-phenol substrate, APEX2 generates biotin-phenoxyl radicals that covalently tag proximal endogenous proteins. Cells are then lysed, and biotinylated proteins are enriched with streptavidin beads and identified by mass spectrometry. We describe the generation of an appropriate APEX2 fusion construct, proteomic sample preparation, and mass spectrometric data acquisition and analysis. A two-state stable isotope labeling by amino acids in cell culture (SILAC) protocol is used for proteomic mapping of membrane-enclosed cellular compartments from which APEX2-generated biotin-phenoxyl radicals cannot escape. For mapping of open cellular regions, we instead use a 'ratiometric' three-state SILAC protocol for high spatial specificity. Isotopic labeling of proteins takes 5–7 cell doublings. Generation of the biotinylated proteomic sample takes 1 d, acquiring the mass spectrometric data takes 2–5 d and analysis of the data to obtain the final proteomic list takes 1 week.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
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DOI of Published Version
https://doi.org/10.1038/nprot.2016.018
