Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans Artan, Murat ; https://orcid.org/0000-0001-8945-6992 Hartl, Markus Chen, Weiqiang De Bono, Mario ; https://orcid.org/0000-0001-8347-0443 ddc:570 Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for Caenorhabditis elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a cofactor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase, and MCCC-1/methylcrotonyl-CoA carboxylase alpha. Here, we developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography. To demonstrate the method's efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, including UNC-10/RIM, SYD-2/liprin-alpha, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, as well as previously uncharacterized potentially synaptic proteins such as the ortholog of human angiomotin, F59C12.3 and the uncharacterized protein R148.3. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin-dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest. Elsevier 2022 info:eu-repo/semantics/article doc-type:article text http://purl.org/coar/resource_type/c_2df8fbb1 https://research-explorer.ista.ac.at/record/12082 https://research-explorer.ista.ac.at/download/12082/12092 Artan M, Hartl M, Chen W, de Bono M. Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans. <i>Journal of Biological Chemistry</i>. 2022;298(9). doi:<a href="https://doi.org/10.1016/j.jbc.2022.102343">10.1016/j.jbc.2022.102343</a> eng info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jbc.2022.102343 info:eu-repo/semantics/altIdentifier/issn/0021-9258 info:eu-repo/semantics/altIdentifier/e-issn/1083-351X info:eu-repo/semantics/altIdentifier/wos/000884241800011 info:eu-repo/semantics/altIdentifier/pmid/35933017 https://creativecommons.org/licenses/by/4.0/ info:eu-repo/semantics/openAccess