The characterization of protein–protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity biotinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches.

蛋白质-蛋白质相互作用 (PPI) 的表征对于理解蛋白质功能具有很高的价值。两种直接从细胞环境中识别 PPI 的策略很流行：亲和捕获（下拉）使用固定化基质分离感兴趣的蛋白质，该基质专门捕获目标和潜在伙伴，而在 BioID 中，生物素连接酶的基因融合促进邻近生物素化，标记的蛋白质用链霉亲和素分离。虽然这两种方法都提供了有价值的见解，但它们可以揭示不同的 PPI，但这些差异的基础并不那么明显。在这里，我们比较了使用四种不同的锥虫蛋白作为诱饵的两种方法：聚 (A) 结合蛋白 PABP1 和 PABP2、mRNA 输出受体 MEX67 和核孔蛋白 NUP158。有了 BioID，我们发现候选相互作用蛋白的数量随着更受限的诱饵蛋白定位而减少，但候选群体随着亲和捕获的变化较小。BioID 更可能返回误报，特别是对于局限性较小的蛋白质，并且识别低分子量蛋白质的效率较低。令人惊讶的是，MEX67 的 BioID 仅鉴定了核孔复合体 (NPC) 内部通道内衬的蛋白质，这与 MEX67 的功能一致，而整个 NPC 是通过下拉分离的。类似地，对于 NUP158，BioID 在 NPC 外环内返回的 PPI 出乎意料地少，而相比之下，这些 PPI 是通过 pulldown 检测到的，而是返回了大量的核蛋白。