Microscopy and proteomics have both revolutionized our understanding of cell biology: while microscopy provides precise spatial information for small numbers of proteins at a time, proteomic methods can detect thousands of proteins but lack spatial information within cells. It would be transformative to combine the strengths of these two fields, to generate an image of the cell in which the complete molecular composition of every spatial region is defined. Such information would represent a quantum leap in our molecular understanding of cellular function, but is beyond the reach of any current technology. This proposal describes a transformative new technology to bridge proteomics and microscopy, to produce the first spatially-resolved proteomic maps of living cells. The key innovation is a nonspecific labeling enzyme that we can genetically target to any region of interest within live cells. Once targeted, we add a chemical substrate to the cell that is converted by the enzyme into a short-lived and highly reactive molecule that chemically labels any protein in its immediate vicinity. Once tagged, the labeled proteins can be isolated and identified by conventional mass spectrometry. Because we know precisely where in the cell the nonspecific labeling enzyme was targeted (e.g. the synaptic cleft), and because the enzyme-generated reactive molecule has a very small labeling radius, any chemically labeled protein that we detect must reside in the vicinity of the nonspecific enzyme (e.g., in the synaptic cleft in this example). We propose to use this technology to map the complete protein composition of many subcellular regions, focusing particularly on those which are poorly understood at the molecular level - such as the synaptic cleft, the mitochondrial inter-membrane space, and organelle-organelle contact zones. In addition to advancing basic molecular and cell biology, this project has many potential medical applications, such as analysis of patient-derived cells and their responses to therapeutics. Such analysis could shed light on the molecular mechanisms of both disease and drug, using only a small fraction of the cellular material required for current proteomic studies. Just as genomic medicine is now revolutionizing medical care, we envision that this project will open the door to proteomic medicine that will provide a critical new layer of information regarding the function/dysfunction of important biological processes in patients.

Public Health Relevance

This project will dramatically enhance our understanding of the inner workings of the cell by providing a complete 'parts list' for different sub-cellular regions, thus enabling far more rapid and comprehensive understanding of their specialized functions. Secondly, this breakthrough technology will lead to fundamentally new methods for diagnosing patients, understanding the natures of their diseases, screening for novel therapeutics, and evaluating the efficacy of such therapeutics. Just as 'genomic medicine' is now revolutionizing medical care, this project will open the door to 'proteomic medicine' that will provide a critical new layer of information regarding the function/dysfunction of important biological processes in patients.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Knowlton, John R
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Stanford University
Schools of Medicine
United States
Zip Code
Branon, Tess C; Bosch, Justin A; Sanchez, Ariana D et al. (2018) Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol 36:880-887
Han, Shuo; Li, Jiefu; Ting, Alice Y (2018) Proximity labeling: spatially resolved proteomic mapping for neurobiology. Curr Opin Neurobiol 50:17-23
Udeshi, Namrata D; Pedram, Kayvon; Svinkina, Tanya et al. (2017) Antibodies to biotin enable large-scale detection of biotinylation sites on proteins. Nat Methods 14:1167-1170
Martell, Jeffrey D; Deerinck, Thomas J; Lam, Stephanie S et al. (2017) Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells. Nat Protoc 12:1792-1816
Han, Shuo; Udeshi, Namrata D; Deerinck, Thomas J et al. (2017) Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells. Cell Chem Biol 24:404-414
Hung, Victoria; Lam, Stephanie S; Udeshi, Namrata D et al. (2017) Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation. Elife 6:
Kaewsapsak, Pornchai; Shechner, David Michael; Mallard, William et al. (2017) Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking. Elife 6:
Lobingier, Braden T; Hüttenhain, Ruth; Eichel, Kelsie et al. (2017) An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells. Cell 169:350-360.e12
Loh, Ken H; Stawski, Philipp S; Draycott, Austin S et al. (2016) Proteomic Analysis of Unbounded Cellular Compartments: Synaptic Clefts. Cell 166:1295-1307.e21
Hung, Victoria; Udeshi, Namrata D; Lam, Stephanie S et al. (2016) Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat Protoc 11:456-75

Showing the most recent 10 out of 12 publications