Objectives This study aims to provide insights into the molecular mechanisms underlying adaptation of CHO-K1 cells to growth in glutamine-free media and potentially identifying critical signalling proteins and pathways involved in this phenotype. Results A CHO-K1 cell line adapted to growth in glutamine-free media was established using a straightforward one-step glutamine reduction strategy. The adapted cell line had a comparable phenotype to the parental cells in terms of cell growth and viability. Global quantitative proteomic and phosphoproteomic analysis was carried out to compare the cells adapted to growth in glutamine-free media to parental cells grown in media containing 8 mM l -glutamine. The adaptation process was accompanied by changes in proteins associated with cytoskeleton rearrangement and mRNA splicing as evidenced via functional analysis of 194 differentially expressed proteins between the two cell lines. 434 phosphoproteins with altered abundance were also identified as a result of adaptation to l -glutamine-free conditions with an associated enrichment of pathways associated with MAPK and calcium signalling. Conclusions This work provides a comprehensive proteomic and phosphoproteomic analysis of protein expression changes after adaptation to glutamine-free growth conditions highlighting critical pathways to consider in the rational design of improved feeding strategies or in cell line engineering to improve bioprocess phenotypes.

中文翻译：

---

适合在无谷氨酰胺培养基中生长的 CHO-K1 细胞的基于 LC-MS/MS 的定量蛋白质组学和磷酸蛋白质组学分析

目的 本研究旨在深入了解 CHO-K1 细胞适应无谷氨酰胺培养基中生长的分子机制，并可能识别与该表型相关的关键信号蛋白和通路。结果 使用简单的一步谷氨酰胺还原策略建立了适合在无谷氨酰胺培养基中生长的 CHO-K1 细胞系。适应细胞系在细胞生长和活力方面具有与亲本细胞相当的表型。进行全局定量蛋白质组学和磷酸蛋白质组学分析以比较适合在无谷氨酰胺培养基中生长的细胞与在含有 8 mM l-谷氨酰胺的培养基中生长的亲本细胞。适应过程伴随着与细胞骨架重排和 mRNA 剪接相关的蛋白质的变化，如通过对两种细胞系之间 194 种差异表达蛋白质的功能分析所证明的那样。还鉴定了 434 种丰度改变的磷蛋白，这是适应无 l-谷氨酰胺条件的结果，伴随着与 MAPK 和钙信号传导相关的途径的相关富集。结论 这项工作提供了对适应无谷氨酰胺生长条件后蛋白质表达变化的全面蛋白质组学和磷酸蛋白质组学分析，突出了在改进喂养策略的合理设计或细胞系工程中要考虑的关键途径，以改善生物过程表型。还鉴定了 434 种丰度改变的磷蛋白，这是适应无 l-谷氨酰胺条件的结果，与 MAPK 和钙信号传导相关的通路富集。结论 这项工作提供了对适应无谷氨酰胺生长条件后蛋白质表达变化的全面蛋白质组学和磷酸蛋白质组学分析，突出了在改进喂养策略的合理设计或细胞系工程中要考虑的关键途径，以改善生物过程表型。还鉴定了 434 种丰度改变的磷蛋白，这是适应无 l-谷氨酰胺条件的结果，伴随着与 MAPK 和钙信号传导相关的途径的相关富集。结论 这项工作提供了对适应无谷氨酰胺生长条件后蛋白质表达变化的全面蛋白质组学和磷酸蛋白质组学分析，突出了在改进喂养策略的合理设计或细胞系工程中要考虑的关键途径，以改善生物过程表型。