International School for Advanced Studies, Trieste, Italy.
Nano Life Science Institute, Kanazawa Medical University, Kanazawa, Japan.
Elife. 2022 Sep 12;11:e77427. doi: 10.7554/eLife.77427.
Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscopy (AFM) to apply a force able to unfold a single protein. The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, which involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from mass spectrometry and proteomic databases. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We validated our pipeline by overexpressing four constructs, and this allowed us to gather structural insights of the identified proteins, revealing variable elements in the loop regions. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment.
单分子力谱（SMFS）使用原子力显微镜（AFM）的悬臂尖端施加能够使单个蛋白质展开的力。所获得的力-距离曲线编码了展开途径，并且可以从其分析中表征折叠结构域。SMFS 主要用于研究溶液中或在脂质双层中重组的纯化蛋白质的展开。在这里，我们描述了一种基于 SMFS 的分析膜蛋白的工作流程，该流程涉及从单个细胞中分离质膜，并直接从质膜中收获力-距离曲线。我们将获得的曲线形状信息与质谱和蛋白质组数据库中的信息结合起来，对嵌入的膜蛋白进行了特征描述和鉴定。该工作流程已通过纯化/重组蛋白进行了测试，并应用于五种细胞类型，对其最丰富的膜蛋白的展开进行了分类。我们通过过表达四个构建体来验证我们的工作流程，这使我们能够收集鉴定出的蛋白质的结构见解，揭示了环区中的可变元素。我们的结果为研究原位膜蛋白的展开以及从膜片段进行蛋白质组学研究奠定了基础。
