Characterization of the Secretome of a Specific Cell Expressing Mutant Methionyl-tRNA Synthetase in Co-Culture Using Click Chemistry

International Journal of Molecular Sciences

Yunsil Chang¹  Sungho Shin²  Narae Park²  Cheolju Lee²  Shinyeong Ju²  Seonjeong Lee²  Yumi Kwon²  Chulwon Ha³  Sunyoung Choi³  Jaehoon Jeong⁴  Kangsik Park⁵ 

[1] Cell and Gene Therapy Institute, Samsung Medical Center, Seoul 06351, Korea;Chemical & Biological Integrative Research Center, Korea Institute of Science and Technology, Seoul 02792, Korea;Department of Orthopedic Surgery, Samsung Medical Center, School of Medicine, Sungkyunkwan University, Seoul 06351, Korea;Division of Radiation Biomedical Research, Korea Institute of Radiological and Medical Sciences, Seoul 01812, Korea;KHU-KIST Department of Converging Science and Technology, Graduate School, Kyung Hee University, Seoul 02447, Korea;

关键词: co-culture;    secretome;    azidonorleucine;    click chemistry;    BONCAT;    mesenchymal stromal cells;   

DOI  :  10.3390/ijms23126527

来源: DOAJ

【 摘 要 】

Co-culture system, in which two or more distinct cell types are cultured together, is advantageous in that it can mimic the environment of the in vivo niche of the cells. In this study, we presented a strategy to analyze the secretome of a specific cell type under the co-culture condition in serum-supplemented media. For the cell-specific secretome analysis, we expressed the mouse mutant methionyl-tRNA synthetase for the incorporation of the non-canonical amino acid, azidonorleucine into the newly synthesized proteins in cells of which the secretome is targeted. The azidonorleucine-tagged secretome could be enriched, based on click chemistry, and distinguished from any other contaminating proteins, either from the cell culture media or the other cells co-cultured with the cells of interest. In order to have more reliable true-positive identifications of cell-specific secretory bodies, we established criteria to exclude any identified human peptide matched to bovine proteins. As a result, we identified a maximum of 719 secreted proteins in the secretome analysis under this co-culture condition. Last, we applied this platform to profile the secretome of mesenchymal stem cells and predicted its therapeutic potential on osteoarthritis based on secretome analysis.

【 授权许可】

Unknown