Cyanobacteria possess unique intracellular organization. Many proteomic studies have examined different features of cyanobacteria to learn about the intracellular structures and their respective functions. While these studies have made great progress in understanding cyanobacterial physiology, the conventional fractionation methods used to purify cellular structures have limitations; specifically, certain regions of cells cannot be purified with existing fractionation methods. Proximity-based proteomics techniques were developed to overcome the limitations of biochemical fractionation for proteomics. Proximity-based proteomics relies on spatiotemporal protein labeling followed by mass spectrometry of the labeled proteins to determine the proteome of the region of interest. We performed proximity-based proteomics in the cyanobacterium Synechococcus sp. PCC 7002 with the APEX2 enzyme, an engineered ascorbate peroxidase. We determined the proteome of the thylakoid lumen, a region of the cell that has remained challenging to study with existing methods, using a translational fusion between APEX2 and PsbU, a lumenal subunit of photosystem II. Our results demonstrate the power of APEX2 as a tool to study the cell biology of intracellular features and processes, including photosystem II assembly in cyanobacteria, with enhanced spatiotemporal resolution.

蓝藻具有独特的细胞内组织。许多蛋白质组学研究已经检查了蓝藻的不同特征，以了解细胞内结构及其各自的功能。虽然这些研究在理解蓝藻生理学方面取得了很大进展，但用于纯化细胞结构的常规分离方法仍有局限性；具体而言，某些细胞区域无法使用现有的分级方法进行纯化。开发了基于邻近的蛋白质组学技术，以克服蛋白质组学生化分离的局限性。基于邻近的蛋白质组学依赖于时空蛋白质标记，然后是标记蛋白质的质谱来确定感兴趣区域的蛋白质组。我们在蓝藻中进行了基于邻近的蛋白质组学聚球藻属 PCC 7002 与 APEX2 酶，一种工程抗坏血酸过氧化物酶。我们使用 APEX2 和 PsbU（光系统 II 的管腔亚基）之间的平移融合，确定了类囊体腔的蛋白质组，这是一个用现有方法研究仍然具有挑战性的细胞区域。我们的结果证明了 APEX2 作为研究细胞内特征和过程的细胞生物学的工具的力量，包括蓝藻中的光系统 II 组装，具有增强的时空分辨率。