Microscopy and mass spectrometry (MS) are complementary techniques: the former provides spatiotemporal information in living cells, but only for a handful of recombinant proteins, while the latter can detect thousands of endogenous proteins simultaneously, but only in lysed samples. Here we introduce technology that combines these strengths by offering spatially- and temporally-resolved proteomic maps of endogenous proteins within living cells. The method relies on a genetically-targetable peroxidase enzyme that biotinylates nearby proteins, which are subsequently purified and identified by MS. We used this approach to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. The labeling was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space (IMS). Several proteins previously thought to reside in the IMS or outer membrane, including protoporphyrinogen oxidase, were reassigned to the matrix. The specificity of live-cell peroxidase-mediated proteomic mapping combined with its ease of use offers biologists a powerful tool for understanding the molecular composition of living cells.

In 1873, Ernst Abbe discovered that features closer than approximately 200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1-5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.

Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments1 or require light and are difficult to use2. Here we report the development of a simple and robust EM genetic tag, called “APEX,” that is active in all cellular compartments and does not require light. APEX is a monomeric 28 kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins. We also fused APEX to the N- or C-terminus of the mitochondrial calcium uniporter (MCU), a newly identified channel whose topology is disputed3,4. MCU-APEX and APEX-MCU give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N-and C-termini of MCU face the matrix.

Summary Obtaining complete protein inventories for subcellular regions is a challenge that often limits our understanding of cellular function, especially for regions that are impossible to purify and are therefore inaccessible to traditional proteomic analysis. We recently developed a method to map proteomes in living cells with an engineered peroxidase (APEX) that bypasses the need for organellar purification when applied to membrane-bound compartments; however, it lacked specificity when applied to unbounded regions that allow APEX-generated radicals to escape. Here, we combine APEX technology with a SILAC-based ratiometric tagging strategy to substantially reduce unwanted background and achieve nanometer spatial resolution. This is applied to map the proteome of the mitochondrial intermembrane space (IMS), which can freely exchange small molecules with the cytosol. Our IMS proteome of 127 proteins has >94% specificity and includes nine novel mitochondrial proteins. This approach will enable scientists to map proteomes of cellular regions that were previously inaccessible.