Development of a Quantitative SRM-Based Proteomics Method to Study Iron Metabolism of <i>Synechocystis</i> sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) is a well-established model species in oxygenic photosynthesis research and a potential host for biotechnological applications. Despite recent advances in genome sequencing and microarray techniques applied in systems biology, quantitative proteomics approaches with corresponding accuracy and depth are scarce for S. 6803. In this study, we developed a protocol to screen changes in the expression of 106 proteins representing central metabolic pathways in S. 6803 with a targeted mass spectrometry method, selected reaction monitoring (SRM). We evaluated the response to the exposure of both short- and long-term iron deprivation. The experimental setup enabled the relative quantification of 96 proteins, with 87 and 92 proteins showing adjusted p-values <0.01 under short- and long-term iron deficiency, respectively. The high sensitivity of the SRM method for S. 6803 was demonstrated by providing quantitative data for altogether 64 proteins that previously could not be detected with the classical data-dependent MS approach under similar conditions. This highlights the effectiveness of SRM for quantification and extends the analytical capability to low-abundance proteins in unfractionated samples of S. 6803. The SRM assays and other generated information are now publicly available via PASSEL and Panorama.集胞藻属（Synechocystis sp.）PCC 6803（以下简称S. 6803）是产氧光合作用研究领域广受认可的模式物种，同时亦是生物技术应用的潜在宿主。尽管当前系统生物学领域的基因组测序与微阵列技术已取得显著进展，但针对S. 6803的高精度、高覆盖度定量蛋白质组学研究仍较为稀缺。本研究采用靶向质谱方法——选择性反应监测（selected reaction monitoring, SRM），建立了可用于检测S. 6803中代表核心代谢通路的106种蛋白质表达变化的实验方案，并评估了该菌株对短期与长期缺铁胁迫的响应。实验体系可实现96种蛋白质的相对定量，其中在短期缺铁与长期缺铁条件下，分别有87种、92种蛋白质的校正后p值小于0.01。本研究通过为共计64种此前在相似实验条件下无法通过经典数据依赖性质谱法检测到的蛋白质提供定量数据，验证了SRM方法针对S. 6803的高灵敏度。该结果凸显了SRM用于蛋白质定量的有效性，同时将S. 6803未分级样品中的低丰度蛋白质分析能力拓展至新的范畴。目前，本研究建立的SRM检测体系与其他生成的相关信息已通过PASSEL与Panorama数据库公开。