Combination of untargeted and targeted proteomics for secretome analysis of L-WRN cells

• Published in: Analytical and bioanalytical chemistry 2023-03, Vol.415 (8), p.1465-1476
• Main Authors: Chen, Zixing, Leung, Thomas Chun Ning, Lui, Ying Lam, Ngai, Sai Ming, Chung, Hau Yin
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Animals; Biochemistry; Cell culture; Cell Line; Cell lines; Cellular proteins; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chromatography, Liquid; Food Science; freeze drying; Growth factors; intestines; Laboratory Medicine; Liquid chromatography; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Mice; Monitoring/Environmental Analysis; Noggin protein; Organoids; Physiological aspects; Proteins; Proteomics; Proteomics - methods; Quality control; Research Paper; Scientific imaging; Secretome; Stability analysis; tandem mass spectrometry; Trichloroacetic acid; Werner Syndrome Helicase; wnt proteins
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-023-04534-9

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, R­spondin3, 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. Graphical Abstract