article published yes Murat Artan author C407B586-6052-11E9-B3AE-7006E66974250000-0001-8945-6992 Stephen Barratt author 57740d2b-2a88-11ec-97cf-d9e6d1b39677 Sean M. Flynn author Farida Begum author Mark Skehel author Armel Nicolas author 2A103192-F248-11E8-B48F-1D18A9856A87 Mario De Bono author 4E3FF80E-F248-11E8-B48F-1D18A9856A870000-0001-8347-0443 MaDe department LifeSc department ISTplus - Postdoctoral Fellowships project Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin's high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method's sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism. https://research-explorer.ista.ac.at/download/10117/10121/2021_JBC_Artan.pdf application/pdfno https://creativecommons.org/licenses/by/4.0/ Elsevier2021 eng 0021-9258 1083-351X 00070640920000610.1016/J.JBC.2021.101094 2973 M. Artan, S. Barratt, S.M. Flynn, F. Begum, M. Skehel, A. Nicolas, M. de Bono, Journal of Biological Chemistry 297 (2021). Artan, M., Barratt, S., Flynn, S. M., Begum, F., Skehel, M., Nicolas, A., &#38; de Bono, M. (2021). Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling. <i>Journal of Biological Chemistry</i>. Elsevier. <a href="https://doi.org/10.1016/J.JBC.2021.101094">https://doi.org/10.1016/J.JBC.2021.101094</a> Artan M, Barratt S, Flynn SM, Begum F, Skehel M, Nicolas A, de Bono M. 2021. Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling. Journal of Biological Chemistry. 297(3), 101094. Artan, Murat, et al. “Interactome Analysis of Caenorhabditis Elegans Synapses by TurboID-Based Proximity Labeling.” <i>Journal of Biological Chemistry</i>, vol. 297, no. 3, 101094, Elsevier, 2021, doi:<a href="https://doi.org/10.1016/J.JBC.2021.101094">10.1016/J.JBC.2021.101094</a>. Artan, Murat, Stephen Barratt, Sean M. Flynn, Farida Begum, Mark Skehel, Armel Nicolas, and Mario de Bono. “Interactome Analysis of Caenorhabditis Elegans Synapses by TurboID-Based Proximity Labeling.” <i>Journal of Biological Chemistry</i>. Elsevier, 2021. <a href="https://doi.org/10.1016/J.JBC.2021.101094">https://doi.org/10.1016/J.JBC.2021.101094</a>. M. Artan <i>et al.</i>, “Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling,” <i>Journal of Biological Chemistry</i>, vol. 297, no. 3. Elsevier, 2021. Artan M, Barratt S, Flynn SM, et al. Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling. <i>Journal of Biological Chemistry</i>. 2021;297(3). doi:<a href="https://doi.org/10.1016/J.JBC.2021.101094">10.1016/J.JBC.2021.101094</a> 101172021-10-10T22:01:23Z2025-04-14T07:43:46Z