Chen Zixing, Leung Thomas Chun Ning, Lui Ying Lam, Ngai Sai Ming, Chung Hau Yin
Food and Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China.
State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China.
Anal Bioanal Chem. 2023 Mar;415(8):1465-1476. doi: 10.1007/s00216-023-04534-9. Epub 2023 Jan 19.
Organoid culture is a promising biomedical technology that requires specialized growth factors. Recently, a recombinant L-WRN cell line has been extensively used to generate conditioned medium (L-CM) for organoid culture. Nevertheless, methods for evaluating the stability of the L-WRN cells have been limited. In this study, a novel proteomics-based approach was developed to analyze the secretome of the cells. Serum-free L-CM was lyophilized, precipitated by trichloroacetic acid, and desalted prior to analysis by liquid chromatography-tandem mass spectrometry. Data-dependent acquisition (DDA) was conducted for the untargeted secretome profiling of the cells, and parallel reaction monitoring (PRM) was applied for the targeted quantification of the Wnt3A, R-spondin3, and noggin proteins (WRNs). This study also compared the performance of two types of PRM methods, namely MS1-independent PRM and MS1-dependent PRM, that can be executed on an Orbitrap instrument. The results showed that the growth of mouse intestinal organoids was closely related to the use of L-CM. The composition of L-CM could be markedly affected by the medium collection scheme. A total of 1725, 2302, and 2681 proteins were identified from the L-CM collected on day 5, day 9, and day 13, respectively. The MS1-independent PRM outperformed the MS1-dependent PRM and effectively quantified the WRNs with high repeatability and specificity. In conclusion, by integrating untargeted and targeted proteomics, this study develops a mass spectrometry-based method for the secretome analysis and quality control of the L-WRN cells. The methodology and findings of the present work will benefit future studies on organoids and secretomes.
类器官培养是一种很有前景的生物医学技术，需要特定的生长因子。最近，重组L-WRN细胞系已被广泛用于生成用于类器官培养的条件培养基（L-CM）。然而，评估L-WRN细胞稳定性的方法一直很有限。在本研究中，开发了一种基于蛋白质组学的新方法来分析细胞的分泌组。无血清L-CM经冻干、三氯乙酸沉淀和脱盐处理后，再通过液相色谱-串联质谱进行分析。对细胞进行数据依赖型采集（DDA）以进行非靶向分泌组分析，并应用平行反应监测（PRM）对Wnt3A、R-spondin3和头蛋白（WRNs）进行靶向定量。本研究还比较了两种可在Orbitrap仪器上执行的PRM方法，即非MS1依赖型PRM和MS1依赖型PRM的性能。结果表明，小鼠肠道类器官的生长与L-CM的使用密切相关。L-CM的组成可能会受到培养基收集方案明显影响。分别从第5天、第9天和第13天收集的L-CM中鉴定出1725、2302和2681种蛋白质。非MS1依赖型PRM的性能优于MS1依赖型PRM，能以高重复性和特异性有效地对WRNs进行定量。总之，通过整合非靶向和靶向蛋白质组学，本研究开发了一种基于质谱的方法用于L-WRN细胞的分泌组分析和质量控制。本工作的方法和研究结果将有助于未来关于类器官和分泌组的研究。
