Introduction: 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. Labeo rohita (rohu) is a fish with a significant role in the global aquaculture economy. Methods: 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. Experiment: 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 (L. rohita) 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. Results: 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. Discussion: This protocol presents new insights on ways to optimize discovery and targeted proteomic analyses of liver tissue using the fish L. rohita 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.
Keywords: ecology; environmental monitoring; fish proteomics; liver proteomics; mass spectrometry; rohu.