--- res: bibo_abstract: - 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.@eng bibo_authorlist: - foaf_Person: foaf_givenName: Murat foaf_name: Artan, Murat foaf_surname: Artan foaf_workInfoHomepage: http://www.librecat.org/personId=C407B586-6052-11E9-B3AE-7006E6697425 - foaf_Person: foaf_givenName: Markus foaf_name: Hartl, Markus foaf_surname: Hartl - foaf_Person: foaf_givenName: Weiqiang foaf_name: Chen, Weiqiang foaf_surname: Chen - foaf_Person: foaf_givenName: Mario foaf_name: De Bono, Mario foaf_surname: De Bono foaf_workInfoHomepage: http://www.librecat.org/personId=4E3FF80E-F248-11E8-B48F-1D18A9856A87 orcid: 0000-0001-8347-0443 bibo_doi: 10.1016/j.jbc.2022.102343 bibo_issue: '9' bibo_volume: 298 dct_date: 2022^xs_gYear dct_identifier: - UT:000884241800011 dct_isPartOf: - http://id.crossref.org/issn/0021-9258 - http://id.crossref.org/issn/1083-351X dct_language: eng dct_publisher: Elsevier@ dct_title: Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans@ fabio_hasPubmedId: '35933017' ...