title: 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.
authors: Department of Chemistry, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.
authors: Victoria
authors: V
authors: Hung
authors: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors: Namrata D
authors: ND
authors: Udeshi
authors: Department of Chemistry, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.
authors: Stephanie S
authors: SS
authors: Lam
authors: Department of Chemistry, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.
authors: Ken H
authors: KH
authors: Loh
authors: Department of Chemistry, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.
authors: Kurt J
authors: KJ
authors: Cox
authors: Department of Chemistry, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.
authors: Kayvon
authors: K
authors: Pedram
authors: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors: Steven A
authors: SA
authors: Carr
authors: Department of Chemistry, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.|Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors: Alice Y
authors: AY
authors: Ting
mesh_terms: D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry; D064251:Multifunctional Enzymes; D015202:Protein Engineering; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
chemical_list: D064251:Multifunctional Enzymes; D020543:Proteome; D001710:Biotin; D006861:Hydrogen Peroxide; D004720:Endonucleases; C530688:APEX2 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase