New Approaches for Localization of Proteins and Interaction Partners in Cells with High Spatial Resolution

Abstract

Labeling of subproteomes within the living cell using genetically‐encoded peroxidase enzyme (APEX; developed in the lab of Alice Ting, Stanford) together with quantitative liquid chromatography‐mass spectrometry is new technology that targets, labels enriches and identifies proteins in subcellular region of interest. In our initial application to the mitochondrial matrix we demonstrated the high specificity of live‐cell peroxidase‐mediated proteomic mapping and the utility for finding novel, previously unknown mitomatrix proteins (Rhee Science 2013). More recently we have extended this powerful new approach to define the molecular composition of unbounded compartments in living cells (Hung Mol Cell 2014; Hung Nature Protocols 2016). Here we illustrate the application of the technology, and recent advances, to define the proteomes of additional sub‐compartments such as excitatory and inhibitory synapses of living neurons. A limitation of the approach has been that few biotinylated sites are identified, reducing the ability to directly obtain information on protein orientation and topology as this requires identifying the sites of biotinylation and not just the proteins that have been modified. This is a known issue with the use of Streptavidin for protein enrichment. We have now developed a new workflow employing anti‐biotin antibodies that increases the numbers of identified sites by >30‐fold and that has manifold potential for studies of biotinylated peptides in a number of other posttranslational modification contexts (Udeshi Nature Methods 2017). Finally, we will describe new work combining recent advances in proximity labeling, genome targeting, and quantitative mass spectrometry to develop genomic locus proteomics (GloPro), a method able to identify proteins that occupy specific genomic loci.Support or Funding InformationThis work is funded by NCI CPTAC PCC 1U24CA210986‐01 to S.A.C. and a Broad Institute SPARC grant to S.M. Portions of this work were also supported by an HHMI Collaborative Innovation Awardsto S.A.C. (Norbert Perrimon PI).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.