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.

邻近依赖性蛋白质标记提供了一种强大的体内策略来表征特定蛋白质的相互作用组。我们之前使用高活性生物素连接酶 TurboID 优化了秀丽隐杆线虫的邻近标记方案。 TurboID 灵敏度的一个重要限制是存在丰富的内源生物素化蛋白质，这些蛋白质占用了质谱仪的带宽，特别是使用生物素作为辅因子的羧化酶。在秀丽隐杆线虫中，这些包括POD-2/乙酰辅酶A羧化酶α、PCCA-1/丙酰辅酶A羧化酶α、PYC-1/丙酮酸羧化酶和MCCC-1/甲基巴豆酰辅酶A羧化酶α。在这里，我们开发了在链霉亲和素纯化和质谱分析之前去除这些羧化酶的方法，方法是对相应的基因进行改造以添加 C 端 His ₁₀标签。这使得我们能够使用固定金属亲和层析从秀丽隐杆线虫裂解物中去除它们。为了证明该方法的功效，我们用它来扩展突触前活性区蛋白 ELKS-1 的相互作用组图。我们鉴定了许多已知的活性区蛋白，包括 UNC-10/RIM、SYD-2/liprin-alpha、SAD-1/BRSK1、CLA-1/CLArinet、C16E9.2/Sentryn，以及以前未表征的潜在突触蛋白，例如作为人血管动蛋白 F59C12.3 和未表征的蛋白 R148.3 的直系同源物。我们的方法为生物素依赖性邻近标记中常见的污染物问题提供了一种快速且廉价的解决方案。该方法可能适用于其他模型生物，并将能够对感兴趣的蛋白质的相互作用物进行更深入、更完整的分析。