Lozano Clément, Armengaud Jean
Département Médicaments et Technologies pour la Santé (DMTS), SPI, Université Paris-Saclay, CEA, INRAE, Bagnols-sur-Cèze, France.
Methods Mol Biol. 2025;2884:57-69. doi: 10.1007/978-1-0716-4298-6_5.
Next-generation shotgun proteomics is one of the most valuable tools for gaining insight into the function of organisms. By providing a list of peptides and abundance information, proteomics enables the identification of proteins, their quantities, posttranslational modifications, and localization. The most refined shotgun proteomics workflow involves protein extraction, trypsin digestion, ultrahigh-performance liquid chromatography coupled to high-resolution tandem mass spectrometry, and confident assignment of resulting spectra to peptide sequences. In this study, we present a versatile, time- and cost-efficient experimental workflow for protein extraction, digestion, and analysis that can be applied to any type of microorganism. Our experimental procedure exhibits superior sensitivity compared to gel-based protocols and can be used for comparative microbial proteomics to highlight key players that explain phenotypic differences between conditions or for proteotyping new microbial isolates for taxonomic purposes.
下一代鸟枪法蛋白质组学是深入了解生物体功能最有价值的工具之一。通过提供肽列表和丰度信息，蛋白质组学能够鉴定蛋白质、其数量、翻译后修饰和定位。最精细的鸟枪法蛋白质组学工作流程包括蛋白质提取、胰蛋白酶消化、超高效液相色谱与高分辨率串联质谱联用，以及将所得光谱可靠地分配到肽序列。在本研究中，我们提出了一种通用的、省时且经济高效的蛋白质提取、消化和分析实验工作流程，该流程可应用于任何类型的微生物。与基于凝胶的方案相比，我们的实验方法具有更高的灵敏度，可用于比较微生物蛋白质组学，以突出解释不同条件之间表型差异的关键因素，或用于对新的微生物分离株进行蛋白质分型以用于分类目的。
