Program in Molecular Medicine, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.
Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
J Proteome Res. 2024 Apr 5;23(4):1531-1543. doi: 10.1021/acs.jproteome.3c00908. Epub 2024 Mar 20.
Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein of interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes and streptavidin resins influence PDB results. We compared the high-confidence interactors of the DNA/RNA-binding protein transactive response DNA-binding protein 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. We also evaluated two commercial affinity resins for purification of biotinylated proteins: conventional streptavidin sepharose versus a new trypsin-resistant streptavidin conjugated to magnetic resin, which significantly reduces the level of contamination by streptavidin peptides following on-bead trypsin digestion. Downstream analyses involved liquid chromatography coupled to mass spectrometry in data-dependent acquisition mode, database searching, and statistical analysis of high-confidence interactors using SAINTexpress. The APEX2-TDP-43 experiment identified more interactors than miniTurbo-TDP-43, although miniTurbo provided greater overlap with previously documented TDP-43 interactors. Purifications on sepharose resin yielded more interactors than magnetic resin in small-scale experiments using a range of magnetic resin volumes. We suggest that resin-specific background protein binding profiles and different lysate-to-resin ratios cumulatively affect the distributions of prey protein abundance in experimental and control samples, which impact statistical confidence scores. Overall, we highlight key experimental variables to consider for the empirical optimization of PDB experiments.
邻近依赖性生物素化 (PDB) 技术可提供有关目标蛋白质分子邻近环境的信息，从而深入了解其功能和定位。在此，我们评估了不同的标记酶和链霉亲和素树脂如何影响 PDB 结果。我们比较了使用 miniTurbo（生物素连接酶）或 APEX2（过氧化物酶）酶鉴定的 DNA/RNA 结合蛋白转录激活反应 DNA 结合蛋白 43kDa（TDP-43）的高可信度相互作用蛋白。我们还评估了两种用于纯化生物素化蛋白质的商业亲和树脂：传统链霉亲和素琼脂糖与新的抗胰蛋白酶的链霉亲和素偶联磁珠，这大大减少了珠上胰蛋白酶消化后链霉亲和素肽的污染水平。下游分析涉及在数据依赖采集模式下进行液相色谱-质谱联用、数据库搜索以及使用 SAINTexpress 对高可信度相互作用蛋白进行统计分析。APEX2-TDP-43 实验比 miniTurbo-TDP-43 鉴定出更多的相互作用蛋白，尽管 miniTurbo 与之前记录的 TDP-43 相互作用蛋白有更大的重叠。在使用一系列磁珠体积的小规模实验中，琼脂糖树脂的纯化比磁珠树脂获得更多的相互作用蛋白。我们认为，树脂特异性背景蛋白结合谱和不同的裂解液-树脂比会累积影响实验和对照样品中猎物蛋白丰度的分布，从而影响统计置信分数。总的来说，我们强调了在 PDB 实验的经验优化中需要考虑的关键实验变量。
