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Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2

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.

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Figure 1: Live-cell proteomics using APEX.
Figure 2: Sample data showing characterization of APEX localization and activity leading up to a proteomic experiment.
Figure 3: Filtering the mass spectrometric data to obtain a final proteomic list.

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Acknowledgements

We thank H.-W. Rhee, P. Zou and J. Martell for help with EM samples. E. Vasile (Koch Institute Microscopy Core Facility) acquired the EM images. We thank O. Aygun and S. Han for their edits to the manuscript. V.H., S.S.L. and K.J.C. were supported by National Science Foundation Graduate Research Fellowships. S.S.L. was also supported by a National Defense Science and Engineering Graduate Fellowship. Funding was provided by the US National Institutes of Health (NIH R01 CA186568 to A.Y.T.) and the Howard Hughes Medical Institute Collaborative Initiative Award (to A.Y.T. and S.A.C.).

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Contributions

V.H. performed the fluorescence characterization of matrix-APEX labeling and the proteomic data analysis. V.H., N.D.U. and A.Y.T. wrote the paper. All authors edited the paper.

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Correspondence to Alice Y Ting.

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The Massachusetts Institute of Technology has submitted a patent application on this work.

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Hung, V., Udeshi, N., Lam, S. et al. Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat Protoc 11, 456–475 (2016). https://doi.org/10.1038/nprot.2016.018

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