Department of Chemical and Biological Engineering, The University of Sheffield, Mappin Street, Sheffield S1 3JD, U.K.
GBCM Laboratory (EA7528), Conservatoire National des Arts et Métiers, HESAM Université, 2 rue Conté, Paris 75003, France.
J Proteome Res. 2024 Mar 1;23(3):999-1013. doi: 10.1021/acs.jproteome.3c00714. Epub 2024 Feb 14.
The value of synthetic microbial communities in biotechnology is gaining traction due to their ability to undertake more complex metabolic tasks than monocultures. However, a thorough understanding of strain interactions, productivity, and stability is often required to optimize growth and scale up cultivation. Quantitative proteomics can provide valuable insights into how microbial strains adapt to changing conditions in biomanufacturing. However, current workflows and methodologies are not suitable for simple artificial coculture systems where strain ratios are dynamic. Here, we established a workflow for coculture proteomics using an exemplar system containing two members, and . Factors affecting the quantitative accuracy of coculture proteomics were investigated, including peptide physicochemical characteristics such as molecular weight, isoelectric point, hydrophobicity, and dynamic range as well as factors relating to protein identification such as varying proteome size and shared peptides between species. Different quantification methods based on spectral counts and intensity were evaluated at the protein and cell level. We propose a new normalization method, named "LFQRatio", to reflect the relative contributions of two distinct cell types emerging from cell ratio changes during cocultivation. LFQRatio can be applied to real coculture proteomics experiments, providing accurate insights into quantitative proteome changes in each strain.
由于能够完成比单一培养物更复杂的代谢任务，合成微生物群落在生物技术中的价值日益凸显。然而，通常需要深入了解菌株间的相互作用、生产力和稳定性，才能优化生长并扩大培养规模。定量蛋白质组学可以深入了解微生物菌株如何适应生物制造中不断变化的条件。然而，目前的工作流程和方法并不适用于菌株比例动态变化的简单人工共培养系统。在这里，我们使用包含两个成员 和 的典型系统建立了共培养蛋白质组学工作流程。研究了影响共培养蛋白质组学定量准确性的因素，包括肽的理化特性，如分子量、等电点、疏水性和动态范围，以及与蛋白质鉴定相关的因素，如不同的蛋白质组大小和种间共享肽。在蛋白质和细胞水平上评估了基于谱计数和强度的不同定量方法。我们提出了一种新的归一化方法，称为“LFQRatio”，以反映在共培养过程中由于细胞比例变化而出现的两种不同细胞类型的相对贡献。LFQRatio 可应用于实际的共培养蛋白质组学实验，为每个菌株的定量蛋白质组变化提供准确的见解。
