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.

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells

“…See our previously published procedure for details 47 . It is essential to confirm that the APEX2 fusion protein exhibits the same localization pattern as the endogenous, untagged protein of interest.…”

“…APEX2 is a multifunctional tag that has been demonstrated for numerous applications beyond EM, including live-cell proteomic mapping 20,21,45–47 , H 2 O 2 -sensing48,49, and fluorescent signal amplification 12,50,51 . The multifunctional capabilities of APEX2 enhance its utility for each of its individual applications.…”

“…We also often use the FLAG epitope tag (DYKDDDDK). Although the V5 and FLAG epitope tags contain lysine residues, which could potentially react with aldehydes during fixation, these tags retain antigenicity when cells are fixed using formaldehyde before immunostaining, following our published procedure 47 . We have not examined whether these epitope tags retain antigenicity after fixation with glutaraldehyde.…”

“…If these criteria are met, it is possible that APEX2 is producing DAB reaction product that cannot be seen by light microscopy but would be detectable by EM. To investigate this possibility, we recommend testing for peroxidase activity using either Amplex UltraRed, as detailed in Box 1, or biotin–phenol followed by fluorescently labeled avidin 47 . These fluorescent readouts provide much more sensitive detection of APEX2 activity, often giving positive signal even when DAB staining is undetectable.…”

“…Formaldehyde fixation, methanol permeabilization, and immunostaining can be performed as described previously 47 . After fixation and permeabilization, bright Amplex UltraRed fluorescence remains trapped inside the cells with a localization pattern matching the subcellular compartment in which APEX2 was expressed.…”