Impact of inherent biases built into proteomic techniques: Proximity labeling and affinity capture compared.
Impact of inherent biases built into proteomic techniques: Proximity labeling and affinity capture compared.
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蛋白质组学技术内置的固有偏见的影响：比较接近性标记和亲和力捕获。
DOI：
10.1016/j.jbc.2022.102726
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发表时间：
2023-01
影响因子：
4.8
通讯作者：
Zoltner, Martin
中科院分区：
文献类型：
--
作者：
Moreira, Claudia Maria do Nascimento;Kelemen, Cristina D.;Obado, Samson O.;Zahedifard, Farnaz;Zhang, Ning;Holetz, Fabiola B.;Gauglitz, Laura;Dallagiovanna, Bruno;Field, Mark C.;Kramer, Susanne;Zoltner, Martin
蛋白质-蛋白质相互作用（PPIs）的表征对理解蛋白质功能具有重要价值。直接从细胞环境中识别PPIs有两种常用的策略：亲和捕获（下拉）用固定基质分离感兴趣的蛋白质，该基质专门捕获目标和潜在的伴侣，而在BioID中，生物素连接酶的遗传融合促进了邻近生物素化，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，但这些差异的基础不太明显。在这里，我们比较了这两种方法，使用四种不同的锥虫蛋白作为诱饵：聚(A)结合蛋白PABP1和PABP2， mRNA输出受体MEX67和核孔蛋白NUP158。使用BioID，我们发现候选相互作用蛋白的数量随着诱饵蛋白定位的限制而减少，但候选群体随着亲和力捕获而变化较小。BioID更有可能返回假阳性，特别是对于定位限制较少的蛋白质，并且识别低分子量蛋白质的效率较低。令人惊讶的是，BioID对MEX67只鉴定了内衬核孔复合物（NPC）内部通道的蛋白质，这与MEX67的功能一致，而整个NPC是通过pull - down分离的。同样，对于NUP158， BioID在NPC外环中返回的PPIs数量少得惊人，而这些PPIs是通过拉下检测到的，相反，BioID返回了更大的核蛋白群。这些相当显著的差异突出了依赖单一方法识别ppi的一个明显问题，并表明BioID和亲和捕获是互补而不是替代方法。
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.
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DOI：
10.1002/wdev.392
发表时间：
2021-01
期刊：
Wiley interdisciplinary reviews. Developmental biology
影响因子：
--
作者：
Bosch JA;Chen CL;Perrimon N
通讯作者：
Perrimon N
锥虫胞外囊肿：一种保守的结构，揭示了内吞作用的新作用。
影响因子：
6.7
作者：
Boehm CM;Obado S;Gadelha C;Kaupisch A;Manna PT;Gould GW;Munson M;Chait BT;Rout MP;Field MC
通讯作者：
Field MC
DOI：
10.1074/mcp.m900038-mcp200
发表时间：
2009-09
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
DeGrasse JA;DuBois KN;Devos D;Siegel TN;Sali A;Field MC;Rout MP;Chait BT
通讯作者：
Chait BT
影响因子：
3.7
作者：
Goos C;Dejung M;Janzen CJ;Butter F;Kramer S
通讯作者：
Kramer S
影响因子：
14.9
作者：
Fritz M;Vanselow J;Sauer N;Lamer S;Goos C;Siegel TN;Subota I;Schlosser A;Carrington M;Kramer S
通讯作者：
Kramer S