Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India.
Central Institute of Fisheries Education, Indian Council of Agricultural Research, Mumbai, Maharashtra, India.
OMICS. 2022 Sep;26(9):489-503. doi: 10.1089/omi.2022.0086. Epub 2022 Aug 26.
The liver is highly sensitive to the environmental factors. Liver tissue, particularly from fish, is often used as a biological target in ecological monitoring, disease research, and stress response studies. (rohu) is a fish with a significant role in the global aquaculture economy. Bottom-up proteomics relies on efficient sample preparation for performing mass spectrometric analysis of the liver tissue. Optimization of protein solubilization and digestion strategies is the key step to obtain reliable data for a successful proteomics experiment. Because the goal of extraction is to acquire the optimum protein quality and yield, the first step should be to choose an appropriate extraction method based on the type of sample. Solubilization buffers containing sodium dodecyl sulfate (SDS) or urea, and digestion methods such as filter-aided sample preparation (FASP), suspension trap (S-Trap) and in-solution are often used in proteomics but are in need of comparative evaluation with an eye to protocol optimization. We applied two different solubilization buffers (one containing SDS, and other containing urea) and three digestion methods (FASP, S-Trap, and in-solution) to the proteomic analysis of the fish () liver tissue. Label-free quantification analysis was performed to analyze the similarities and differences in the results with each method. Gene ontology-based functional analysis was performed for the identified proteome across the experimental conditions to overview their protein classes, molecular functions, and biological processes. SDS lysis followed by S-Trap digestion outperformed the other combinations of lysis and digestion in terms of higher protein coverage, consistency in the results and repeatability. Filter-based methods provided comparatively better results than in-solution digestion. This protocol presents new insights on ways to optimize discovery and targeted proteomic analyses of liver tissue using the fish as a case study. Other tissues can also be evaluated in the future drawing from the results in this study. This would help the scientific community with hypothesis-driven studies on topics ranging from basic biology to applied aquaculture research and ecological monitoring. This is particularly relevant in the current era of ecological crises and environmental pollution, where advances and optimization in research protocols can contribute to in-depth studies of ecosystems and planetary health.
肝脏对外界环境因素高度敏感。肝脏组织，特别是鱼类的肝脏组织，常被用作生态监测、疾病研究和应激反应研究的生物靶标。罗非鱼是全球水产养殖经济中的重要鱼类。自下而上的蛋白质组学依赖于有效的样品制备，以便对肝脏组织进行质谱分析。优化蛋白质溶解和消化策略是获得可靠数据以成功进行蛋白质组学实验的关键步骤。由于提取的目标是获得最佳的蛋白质质量和产量，因此第一步应该根据样品类型选择合适的提取方法。含有十二烷基硫酸钠（SDS）或尿素的溶解缓冲液，以及过滤辅助样品制备（FASP）、悬浮陷阱（S-Trap）和溶液中的消化方法等常用于蛋白质组学，但需要进行比较评估，以优化方案。我们应用了两种不同的溶解缓冲液（一种含有 SDS，另一种含有尿素）和三种消化方法（FASP、S-Trap 和溶液中）对罗非鱼肝脏组织的蛋白质组学分析。进行无标记定量分析，以分析每种方法的结果相似性和差异。对跨实验条件鉴定的蛋白质组进行基于基因本体论的功能分析，以概述其蛋白质类别、分子功能和生物学过程。SDS 裂解后再进行 S-Trap 消化，在蛋白质覆盖率、结果一致性和重复性方面优于其他裂解和消化组合。基于过滤的方法比溶液消化提供了更好的结果。该方案为使用罗非鱼作为案例研究优化肝脏组织的发现和靶向蛋白质组学分析提供了新的见解。未来可以从本研究的结果中评估其他组织。这将有助于科学界进行从基础生物学到应用水产养殖研究和生态监测的各种主题的假设驱动研究。在当前生态危机和环境污染的时代，这一点尤其重要，因为研究方案的进步和优化可以促进对生态系统和行星健康的深入研究。
