使用邻近标记技术（例如基于生物素连接酶的 BioID）进行蛋白质-蛋白质相互作用研究已成为理解细胞过程不可或缺的一部分。大多数研究利用传统的 2D 细胞培养系统，可能忽略了 3D 组织中蛋白质行为的重要差异。在这项研究中，我们研究了蛋白质 Bcl-2 细胞死亡激动剂 (BAD) 的蛋白质-蛋白质相互作用，并将传统 2D 培养条件与 3D 系统进行比较，其中细胞嵌入 3D 细胞外基质 (ECM) 模拟物中。使用与工程化生物素连接酶 miniTurbo (BirA*) 融合的 BAD，我们在 2D 和 3D 条件下鉴定了重叠和不同的 BAD 相互作用组。已知的 BAD 结合蛋白 14-3-3 亚型和 Bcl-XL 在 2D 和 3D 中与 BAD 相互作用。在确定的 131 个 BAD 相互作用因子中，56% 是 2D 特异性的，14% 是 3D 特异性的，30% 是两种情况共有的。相互作用网络分析证明了 2D 和 3D 相互作用组之间的差异关联，强调了培养条件对蛋白质相互作用的影响。 2D-3D 重叠相互作用组封装了细胞凋亡程序，这是 BAD 的众所周知的作用。 3D 独特的通路在 ECM 信号传导中丰富，表明 BAD 具有迄今为止未知的功能。因此，探索 3D 中蛋白质-蛋白质相互作用为细胞行为提供了新的线索。这种令人兴奋的方法有可能弥合易处理的 2D 细胞培养和类器官 3D 系统之间的知识差距。

Protein-protein interaction studies using proximity labeling techniques, such as biotin ligase-based BioID, have become integral in understanding cellular processes. Most studies utilize conventional 2D cell culture systems, potentially missing important differences in protein behavior found in 3D tissues. In this study, we investigated the protein-protein interactions of a protein, Bcl-2 Agonist of cell death (BAD), and compared conventional 2D culture conditions to a 3D system, wherein cells were embedded within a 3D extracellular matrix (ECM) mimic. Using BAD fused to the engineered biotin ligase miniTurbo (BirA*), we identified both overlapping and distinct BAD interactomes under 2D and 3D conditions. The known BAD binding proteins 14-3-3 isoforms and Bcl-XL interacted with BAD in both 2D and 3D. Of the 131 BAD-interactors identified, 56% were specific to 2D, 14% were specific to 3D, and 30% were common to both conditions. Interaction network analysis demonstrated differential associations between 2D and 3D interactomes, emphasizing the impact of the culture conditions on protein interactions. The 2D-3D overlap interactome encapsulated the apoptotic program, which is a well-known role of BAD. The 3D unique pathways were enriched in ECM signaling, suggestive of hitherto unknown functions for BAD. Thus, exploring protein-protein interactions in 3D provides novel clues into cell behavior. This exciting approach has the potential to bridge the knowledge gap between tractable 2D cell culture and organoid-like 3D systems.