Russo David A, Schneidmadel Felix R, Zedler Julie A Z
Bioorganic Analytics, Institute for Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, 07743 Jena, Germany.
Functional Proteomics, Jena University Hospital, 07747 Jena, Germany.
Plant Physiol. 2025 Sep 1;199(1). doi: 10.1093/plphys/kiaf334.
Cyanobacteria have played a leading role in elucidating the fundamental mechanisms behind oxygenic photosynthesis, carbon fixation, the circadian clock, and phototaxis. Such molecular processes rely on proteins at their core. Thus, proteomics has become an indispensable tool in building our understanding of these processes. Amongst the proteomic approaches used, "shotgun proteomics", where complex protein mixtures are enzymatically digested into peptides and analyzed by liquid chromatography-mass spectrometry, has become the go-to technique for whole-proteome analysis. In this study, we introduce shotgun workflows that excel in speed, throughput, and sensitivity, and allow an in-depth description of the cyanobacterial proteome. The main features of these workflows are the improvement of sample cleanup and digestion through single-pot solid phase-enhanced sample preparation (SP3), the adoption of a previously validated trifluoroacetic acid lysis strategy, and the application of library-free data-independent acquisition. Using the established model organism Synechococcus elongatus PCC 7942, we show that these workflows exhibit high quantitative reproducibility and enable the detection of 83% to 85% of all open reading frames, the greatest single-shot coverage achieved so far for a cyanobacterium. These workflows require only a couple of hours of hands-on time and should be applicable to most, if not all, cyanobacterial species. Together with the rapid advancements in mass spectrometry technologies, this work has the potential to accelerate cyanobacterial proteomics.
蓝细菌在阐明产氧光合作用、碳固定、生物钟和趋光性背后的基本机制方面发挥了主导作用。这些分子过程核心依赖于蛋白质。因此，蛋白质组学已成为增进我们对这些过程理解的不可或缺的工具。在所使用的蛋白质组学方法中，“鸟枪法蛋白质组学”，即将复杂蛋白质混合物酶解成肽段并通过液相色谱 - 质谱联用分析，已成为全蛋白质组分析的首选技术。在本研究中，我们介绍了在速度、通量和灵敏度方面表现出色的鸟枪法工作流程，这些流程能够深入描述蓝细菌蛋白质组。这些工作流程的主要特点包括通过单锅固相增强样品制备（SP3）改进样品净化和消化，采用先前验证的三氟乙酸裂解策略，以及应用无文库数据非依赖采集。使用已建立的模式生物聚球藻PCC 7942，我们表明这些工作流程具有高定量重现性，能够检测到所有开放阅读框的83%至85%，这是迄今为止蓝细菌单次获得的最大覆盖率。这些工作流程仅需几个小时的实际操作时间，并且应该适用于大多数（如果不是全部）蓝细菌物种。随着质谱技术的快速发展，这项工作有可能加速蓝细菌蛋白质组学的研究。
