ArticleNature protocols2016
Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2.
Article in Nature protocols, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 363 papers.
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Who cites it
363 citing papers in PubMed, 596 citations in OpenAlex.
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- A modular APEX biosensor enables sensitive and visualized monitoring of viral protease activity.Virologica Sinica · 2026Article
- Spatial architecture of immunometabolism: Mitochondrial‑organelle interfaces in immune signaling (Review).International journal of molecular medicine · 2026Review
- APEX-seq maps transcriptome-wide subcellular RNA localization in living cells.Nature protocols · 2026Review
- An INF2-dependent actin-mediated step in inositol 1,4,5-trisphosphate receptor cluster formation and activity.Current biology : CB · 2026Article
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303 more citing papers are in PubMed but not listed here.
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Authors and funding
8 authors at 2 institutions in 1 country.
Funding
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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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.