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.

Figure 1

Live-cell proteomics using APEX. (a) Scheme showing APEX-catalyzed biotinylation. The mitochondrial matrix is enclosed by the IMM. This example shows APEX (green Pac-Man) targeted to the mitochondrial matrix^,. Live cells are incubated with biotin-phenol probe (red B = biotin) for 30 min and then treated for 1 min with 1 mM H₂O₂ to initiate biotinylation. APEX and APEX2 catalyze the one-electron oxidation of biotin-phenol into a biotin-phenoxyl radical, which covalently tags proximal endogenous proteins. The biotin-phenoxyl radical does not cross the IMM. (b) Two-state SILAC experiment used to map the proteome of the mitochondrial matrix. The experimental sample was cultured in heavy (H) arginine and lysine amino acids. The negative control sample, with APEX omitted, was cultured in light (L) arginine and lysine amino acids. Each dish of cells was separately treated with biotin-phenol and H₂O₂ and then lysed, and streptavidin enrichment was performed on the combined lysates. Subsequent processing steps are shown. For each detected peptide, the H/L intensity ratio reflects the extent to which that peptide was biotinylated by APEX. Nonspecific binders have H/L ratios close to 1. (c) Three-state SILAC experiment used to map the proteome of the IMS. The IMS lies in between the IMM and the OMM, which contains porins that allow the exchange of molecules < 5 kDa (ref. 16). Consequently, biotin-phenoxyl radicals generated by IMS-localized APEX can escape into the cytosol, leading to background (biotinylation contour map outside the OMM shown in drawing). To subtract this background, we used a three-state rather than two-state SILAC experiment. The heavy sample expresses APEX targeted to the IMS^,. The light sample is a nonbiotinylated negative control, just as in b. The medium (M) sample expresses APEX outside the ROI (in this case, the cytosol, via fusion to a nuclear export sequence peptide). Streptavidin enrichment is performed on the mixture of H, M and L lysates, as shown in b. For each MS-detected peptide, the H/L intensity ratio reflects the extent of biotinylation by IMS-APEX. The H/M ratio reflects the extent to which that peptide is preferentially biotinylated by IMS-APEX versus cytosolic APEX. Adapted from Science 339, 1328–1331 (2013). Reprinted with permission from AAAS; and adapted from ref. 5 with permission.

Figure 2

Sample data showing characterization of APEX localization and activity leading up to a proteomic experiment. (a) EM images of cells expressing APEX targeted to the mitochondrial matrix (top) and intermembrane space (bottom). Dark regions indicate the presence of APEX^,,,. Scale bars, 100 nm. The IMS-APEX EM image was reproduced with permission from ref. 5. (b) Confocal fluorescence imaging of matrix-APEX labeling in cells. Human embryonic kidney (HEK) 293T cells were transfected with matrix-APEX and labeled live as in Figure 1a. In parallel, samples in which either H₂O₂ or biotin-phenol (BP) was omitted were prepared as negative controls (rows 2 and 3). Cells were fixed and stained with a NeutrAvidin–Alexa Fluor 647 (AF647) conjugate to visualize biotinylated proteins and anti-V5 antibody to visualize matrix-APEX localization. The untransfected cell in the top row (starred, *) shows that biotinylation is dependent on APEX expression. DIC, differential interference contrast image. Scale bars, 10 μm. (c) Characterization of APEX-mediated biotinylation of endogenous proteins by streptavidin blotting. HEK 293T cells were transfected with IMS-APEX or matrix-APEX ( >50% transfection efficiency) and labeled as in b. Cells were then lysed, separated by SDS-PAGE and analyzed by blotting with streptavidin-horseradish peroxidase (SA-HRP). The right shows Ponceau S staining of the same membrane. Negative controls in which BP, H₂O₂ or APEX was omitted are shown in lanes 2 and 3 of each blot. The IMS-APEX streptavidin-HRP blot was reproduced from ref. 5 with permission. The matrix-APEX streptavidin-HRP blot was reproduced from Science 339, 1328–1331 (2013). Reprinted with permission from AAAS. (d) Streptavidin enrichment of proteins biotinylated by matrix-APEX. Whole-cell lysates were prepared as in c. Biotinylated proteins within lysates were then enriched using streptavidin-coated magnetic beads and eluted by boiling in SDS and biotin and analyzed by SDS-PAGE and Coomassie staining. Negative controls in which APEX was omitted (lane 2) or substrates were omitted (lane 3) are shown. Elution of beads that were not incubated with lysate is shown in lane 4. Reproduced from Science 339, 1328–1331 (2013). Reprinted with permission from AAAS.

Figure 3

Filtering the mass spectrometric data to obtain a final proteomic list. (a) Analysis of two-state SILAC data from the mitochondrial matrix proteomic experiment. In total, 3,430 proteins were identified by three or more unique peptides in replicate 1 of this experiment. To plot the true positive distribution, we took the subset of 808 proteins with prior mitochondrial annotation in the Gene Ontology Cell Component (GOCC) database, MitoCarta or literature (green histogram). The false positive distribution (red histogram) is given by the 521 proteins from this data set that were found in a hand-curated list of 2,410 non-mitochondrial proteins. The green population has a right-shifted distribution compared with the red population, which indicates that the log₂(H/L) ratio allows us to distinguish bona fide mitochondrial proteins from non-mitochondrial proteins. Adapted from Science 339, 1328–1331 (2013). Reprinted with permission from AAAS. (b) Determination of the optimal SILAC ratio cutoff for the data in a. For every possible SILAC cutoff, the true positive rate (TPR) was plotted against the false positive rate (FPR) in a receiver operating characteristic (ROC) curve (top). TPR is defined as the fraction of detected true positive proteins above the cutoff. FPR is defined as the fraction of detected false positive proteins above the cutoff. The bottom graph plots the difference between the TPR and FPR at every SILAC ratio cutoff. The dashed line indicates the log₂(H/L) ratio at which TPR-FPR is maximal. This SILAC ratio is used as our cutoff, and it is depicted in the histogram in a as a dashed line. (c) Analysis of three-state SILAC data from the mitochondrial IMS proteomic experiment. Here, the data are presented as a scatter plot rather than histogram, because two rather than one SILAC ratios are used for the data filtering. In this plot, the log₂(H/M) value is plotted against the log₂(H/L) value for each of the 4,868 proteins detected in this experiment. Known IMS-exposed proteins (true positives) are colored in green, proteins that lack prior mitochondrial annotation (possible false positives) are colored in red and all other proteins are colored in black. The dashed lines indicate the SILAC ratio cutoffs that were calculated via TPR-FPR analysis performed as in b. The inset shows the complete data. Adapted from ref. 5 with permission.